Ly6H gene

ABSTRACT

The invention provides a brain-specific gene useful in treating Alzheimer&#39;s disease, for instance, which comprises a nucleotide sequence cording for the amino acid sequence shown in SQ ID NO:1 and fragments thereof; an expression vector comprising the gene; a host cell comprising the expression vector; an expression product of the gene; an antibody against the product; a therapeutic and prophylactic composition for neurodegenerative disease; and the like.

This Application is a Continuation of U.S. application Ser. No.09/787,360, filed Mar. 16, 2001 now abandoned, which in turn is a 371 ofPCT/JP99/05039, filed Sep. 16, 1999; the disclosure of each of which isincorporated herein by reference.

TECHNICAL FIELD

The invention relates to a gene expressed with high specificity at ahigh level in the brain, more particularly a gene coding for a novelprotein belonging to the Ly6 Family (cf. the literature cited below)which has been utilized in the purification of blood stem cells, studieson the differentiation of blood cells, activation of immune cells,inhibition of production of active immune cells, treatment of tumors andthe like. The invention further relates to a novel protein encoded bysaid gene and to its specific antibody. In addition, the inventionrelates to a therapeutic and prophylactic composition forneurodegenerative disease such as Alzheimer's disease.

BACKGROUND ART

Proteins of the Ly6 family have a low-molecular weight GPI-anchoredstructure and have been identified as a class of cell surfaceglycoproteins forming a gene cluster on mouse chromosome 15 [Proc. Natl.Acad. Sci., USA., 84, 1638-1643 (1987)].

The Ly6 family is specifically expressed at high levels in bone marrowcells and lymphoid cells and, therefore, has been utilized as a markerfor T-cell differentiation and hematopoietic stem cells [Immunol. CellBiol., 73, 277-296 (1995)]. While much remains to be known about itsfunctions in vivo, the finding that its expression is highly modulatedin the lymphocytic system suggests that these proteins are playingimportant roles in the immune system, particularly in thedifferentiation and function of T cells. It is reported that Ly6c, forinstance, mediates the homing of CD8⁺ T cells to the lymph node throughintegrin-dependent adhesion [Proc. Natl. Acad. Sci., USA., 94, 6898-6903(1997)].

Furthermore, many GPI-anchored proteins are known to interact withprotein kinases [Science, 254, 1016-1019 (1991)]. For example, theinteraction of Ly6 with p56lck and p59fyn suggests the likelihood of itsinvolvement in the signal transduction of T cells [Eur. J. Immunol., 23,825-831 (1993)]. It is also reported that T cells derived fromLy6a-defective mice have been enhanced in the ability to proliferate inresponse to antigenic stimulation [J. Exp. Med., 186, 705-717 (1997)].The possibility of its regulating not only the activation of T cells butalso that of B cells has also been suggested [J. Immunol., 144,2197-2204 (1990)].

Furthermore, several GPI-anchored proteins are known to have beenexpressed and be functioning in both the lymphocytic system and thenervous system [Nature, 379, 826-829 (1996); Curr. Biol., 7, 705-708(1997)]. In the Ly6 family, Ly6a.2 and Ly6E are reportedly present andfunctioning in both systems [Proc. Natl. Acad. Sci., USA., 85, 2255-2259(1996); J. Immunol, 157, 969-973 (1996)].

Elucidation of the physiological roles played by such proteins of theLy6 family and the genes coding for the proteins and the resultinginformation are considered to be of use in the field of fundamentalscientific research as well as in the pharmaceutical field in connectionwith the purification of blood stem cells, studies on thedifferentiation of blood cells, activation of immune cells, inhibitionof activation of immune cells, therapy of tumors, and the like.

Recently, in patients with Alzheimer's disease, an excessive cerebraltemperal lobe atrophy as compared with age-associated brain atrophy hasbeen reported [Jobst, K. A., et al., Lancet, 343, 829-830 (1994)],suggesting that some gene or genes having a bearing on the cerebraltemperal lobe are somehow associated with the onset and progression ofAlzheimer's disease. It is logical to assume that should such a gene beidentified or characterized, there might be provided information usefulfor the therapy and prophylaxis of Alzheimer's disease.

Therefore, an object of the present invention is to provide the aboveinformation needed by those concerned, particularly a novel humanprotein belonging to the Ly6 family and a gene coding for the protein.

A further object of the invention is to provide a pharmaceuticalcomposition for the therapy and prophylaxis of various neurodegenerativediseases, represented by Alzheimer's disease.

The present inventor explored into the genes derived from various humantissues and succeeded in isolating and characterizing a novelbrain-specific gene meeting the above objects. The inventor furtherfound that the level of expression of this newly isolated gene ismarkedly depressed in the temperal lobe, inclusive of the hippocampusand entorhinal cortex, of a patient with Alzheimer's disease, that thisis a causative factor in the onset and progression of Alzheimer'sdisease and in dementia and other disturbances and that this gene andits expression product can be exploited with advantage in the therapyand prophylaxis of Alzheimer's disease. The present invention has beenaccomplished on the basis of the above findings.

DISCLOSURE OF INVENTION

The present invention provides a gene comprising a nucleotide sequencecoding for the following protein (a) or (b).

(a) a protein having the amino acid sequence shown in SEQ ID NO:1

(b) a protein having an amino acid sequence derived from the amino acidsequence shown in SEQ ID NO:1 by deletion, substitution or addition ofone or a plurality of amino acids and having at least one physiologicalactivity selected from the group consisting of neuronalsurvival-supporting activity, nerve elongating activity, nerveregenerating activity, neuroglia-activating activity, and mnemonic(brain memory-forming) activity.

The invention also provides the above gene wherein the nucleotidesequence is shown in SEQ ID NO:2, in particular, which is a human gene.

Furthermore, the invention provides a gene comprising the followingpolynucleotides (a) or (b), particularly the corresponding human gene.

(a) a polynucleotide containing the nucleotide sequence shown in SEQ IDNO:3

(b) a polynucleotide which hybridizes under stringent condition with aDNA having the nucleotide sequence shown in SEQ ID NO:3.

The invention further provides a gene expression vector harboring saidgene; a host cell harboring said gene expression vector; an expressionproduct which is expressed by said host cell; a protein encoded by thegene of the invention; and an antibody which binds said expressionproduct or said protein.

The invention further provides a therapeutic and prophylacticcomposition for neurodegenerative disease, which comprises said proteinor an equivalent thereof or said expression product as an activeingredient in combination with a pharmaceutical carrier. Moreparticularly, the invention provides the therapeutic and prophylacticcomposition for neurodegenerative disease, wherein said activeingredient is a protein having the amino acid sequence shown in SEQ IDNO:1 or an equivalent thereof or a gene product obtainable by expressionof the whole or part of a gene comprising a nucleotide sequence shown inSEQ ID NO:2 and having at least one physiological action selected fromthe group consisting of neuronal survival-supporting action, nerveelongating action, nerve-regenerating action, neuroglia-activatingaction, and brain memory-forming (mnemonic, encoding) action.

Especially, the invention provides the therapeutic and prophylacticcomposition for Alzheimer's disease, Alzheimer type dementia, brainischemia and Parkinson's disease.

In addition, the invention provides a sense strand oligonucleotidecomprising at least 20 consecutive constituent nucleotides of thenucleotide sequence shown in SEQ ID NO:2; a gene therapy compositioncomprising said sense strand oligonucleotide as an active ingredient incombination with a pharmaceutical carrier; and a gene-specific probecomprising an oligonucleotide sequence of at least 10 consecutiveconstituent nucleotides of the nucleotide sequence shown in SEQ ID NO:2.

Furthermore, the invention provides a method of screening for candidatecompounds either capable of binding to said protein, equivalent thereofor expression product or influencing its activity which comprises usingsaid protein, equivalent or expression product; a kit for saidscreening; and said compounds so screened.

Representation of amino acids, peptides, nucleotide sequences,nucleotides, etc. by abbreviations in the specification is in conformitywith the rules recommended by IUPAC-IUB [IUPAC-IUB Communication onBiological Nomenclature, Eur. J. Biochem., 138, 9 (1984)], “Guidelinefor drafting patent specifications relative to nucleotide sequencesand/or amino acid sequences” (edited by the Patent Office of Japan) andthe conventions relating to the use of codes or symbols in the art.

A specific example of the gene of the invention is the gene deduced fromthe DNA sequence of the PCR product designated “LY6H” as described inthe example which appears later herein. Its nucleotide sequence is asshown in SEQ ID NO:3.

The LY6H gene is a cDNA containing a 420-codon open reading frame (ORF)coding for a novel brain-specific protein (LY6H protein) having a140-residue amino acid sequence as shown in SEQ ID NO:1, and having afull-length sequence of 854 nucleotides.

The LY6H protein which is the expression product of the gene of theinvention was found to have high homology to mouse Ly6 family proteins[Immunol. Cell Biol., 73, 277-296 (1995)] by a GenBank/EMBL databasesearch using FASTA Program (Person, W. R. et al., Proc. Natl. Acad.Sci., USA., 85, 2444-2448 (1988)). Furthermore, high gene-to-genehomology was recognized. Therefore, the gene of the invention isconsidered to be a novel human Ly6 gene.

The LY6 gene of the invention was identified to be a gene which isspecifically expressed in the brain by the sequencing of more than 28000cDNA clones selected at random from a fetal human brain cDNA library. ByRH chromosome mapping [Hum. Mol. Genet., 5, 339-346 (1996)], the locusof the gene on the chromosome was found to be 8q24. 3.

Thus, the gene and expression product of the invention, thus provided,contribute to detection of the expression of the gene in varioustissues, production of human LY6H protein by genetic engineeringtechniques, and construction of an antibody thereto, hence enabling thepurification of hematopoietic stem cells, study of blood celldifferentiation, activation or suppression of immune cells, therapy oftumors, and the like.

In addition, the expression product (polypeptide) of the invention, thusprovided, enables provision of a drug for prophylaxis and therapy ofneurodegenerative diseases such as Alzheimer's disease, Alzheimer typedementia, Parkinson's disease and ischemic brain. Furthermore, the sensestrand of the gene according to the invention can be utilized as apharmaceutical composition for gene therapy, with which the onset andprogression of the above-mentioned neurodegenerative diseases can beinhibited or arrested.

The invention further provides a method of screening for compoundseither binding or influencing the activity of the expression product(polypeptide) of the invention and a relevant kit for screening, hencecompounds so screened as well. For identification of such compoundsscreened, an antibody binding to the expression product of the gene ofthe invention can be utilized.

In the specification, the term “gene” is used to mean a double-strandedDNA and its constituent single-stranded DNA, whether sense or antisense,without regard to its length. Therefore, unless otherwise indicated, thegene (DNA) of the invention includes a double-stranded DNA containing ahuman genomic DNA, a single-stranded DNA (sense strand) inclusive of thecDNA, a single-stranded DNA (antisense strand) having a sequencecomplementary to said sense strand, and fragments of said DNAs.

The gene (DNA) of the invention may contain a leader sequence, a codingregion, exons and introns. The polynucleotide includes both RNA and DNA.The DNA includes cDNA, genomic DNA and synthetic DNA. The polypeptideincludes its fragments, homologs, derivatives and mutants. The mutantsinclude alleles which occur naturally, mutants not existing naturally,mutants having amino acid sequences mutated by deletion, substitution,addition and/or insertion, and mutants having functionally equivalentmodified amino acid sequences.

Such modifications (e.g. mutations) of amino acid sequences may forexample occur from spontaneous mutation or posttranslationalmodification but may be artificially induced by utilizing a native gene(for example, specific genes of the invention).

The homology of such mutants to the unmutated polypeptide may be atleast 70%, preferably 80%, more preferably 95%, still more preferably97%. The above polypeptide and its mutants and homologs have astructural feature conserved in common and may have the biologicalactivities of the expression product of the gene of the invention, suchas neuronal survival-supporting action, neuronal growth-stimulatingaction, nerve generating action, and neuroglia-stimulating action. Thehomology of polypeptides can be analyzed by searching through a databasesuch as SWISSPLOTS Database using a sequence analysis software such asFASTA [Clustal, V., Methods Mol. Biol., 25, 307-318 (1994)].

The gene coding for such a mutant is silent or conserved for amino acidsubstitution. Thus, the amino acid residues encoded by the nucleotidesequence are not altered.

The conservatively substitutable amino acid residues, i.e. the aminoacid residues substitutable with other amino acid residues withoutlosing the activities of the polypeptide having such original amino acidresidues, and the corresponding original amino acid residues are asfollows.

Original amino Conservatively substituting acid residue amino acidresidue Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp GlyPro His Asn or Gln Ile Leu or Val Leu Ile or Val Lys Arg, Aln or Glu MetLeu or Ile Phe Met, Leu or Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp or PheVal Ile or Leu

In addition, Cys may be substituted for a different kind of amino acidresidue, e.g. Ser, Ala or Val.

The gene and expression product according to the invention provideinformation and means of great use for the elucidation, expatiation,diagnosis, prophylaxis and therapy of neurodegenerative diseases such asAlzheimer's disease, brain ischemia and Parkinson's disease. The gene ofthe invention can also be used with advantage for the development of newdrugs capable of inducing expression of the gene. for use in thetreatment of said neurode-generative diseases. In addition, thedetection of expression of the gene of the invention and of theresulting expression product in individuals or tissues, and thedetection of mutation (deletion or point mutation) of the gene orabnormal expression thereof can be utilized with advantage for theelucidation and diagnosis of said neurodegenerative diseases.

The gene of the invention includes but is not limited to the gene havingthe nucleotide sequence shown in SEQ ID NO:2 which codes for a proteinhaving the amino acid sequence shown in SEQ ID NO:1, for example a gene(LY6H gene) having the nucleotide sequence shown in SEQ ID NO:3. Forexample, the gene of the invention may be a gene coding for an aminoacid sequence derived from the above-defined amino acid sequence by agiven modification, a gene coding for an amino acid sequence having agiven degree of homology to the above-defined amino acid sequence, or agene having a nucleotide sequence having a given degree of homology toany of the above genes.

The above-mentioned given degree of homology to a defined amino acidsequence or nucleotide sequence means a homology of at least not lessthan 70%, preferably not less than 90%, more preferably not less than95%, still more preferably not less than 97%. The present inventionencompasses homologs (gene homologs and protein homologs) having suchhomology.

The gene of the invention includes “a gene coding for a polypeptidehaving an amino acid sequence (modified amino acid sequence) derivedfrom the amino acid sequence shown in SEQ ID NO:1 by deletion,substitution or addition of one or a plurality of amino acids”. Theextent and position or positions of “deletion, substitution or addition”are not particularly restricted insofar as the resulting polypeptidehaving a modified amino acid sequence is equivalent, in biologicalfunction, to the polypeptide (LY6H protein) having the amino acidsequence shown in SEQ ID NO:1. The biological “function” mentioned aboveincludes physiological functions such as neuronal survival-supportingaction, nerve elongating action, nerve regenerating action,neuroglia-activating action, and mnemonic action, and the “equivalent”is a polypeptide having such functions. Therefore, the protein havingsuch a modified amino acid sequence includes a protein (equivalent)having a fragment (a consecutive-residue fraction) of the amino acidsequence shown in SEQ ID NO:1 and having physiological activitiessimilar to those mentioned above for the full-length of said amino acidsequence. Furthermore, the gene coding for a polypeptide having theabove-modified amino acid sequence may be a gene with which the gene ofthe invention encoding a polypeptide having the pre-modification aminoacid sequence can be detected. The term plurality as used in connectionwith said modification usually means not less than 2 but up to several,although the range is not restrictive.

The homolog of LY6H gene (and the homolog of the expression product ofthe gene) according to the invention means any of a series of relatedgenes (and their expression products) which are homologous in sequenceand recognized as one gene family from their structural characteristics,common gene expression pattern, and similarities in said biologicalfunction. This, of course, includes alleles of the genes of theinvention.

The modification (mutation) of an amino acid sequence may occurnaturally, for example by spontaneous mutation and posttranslationalmodification but may be induced artificially on the basis of the nativegene (for example the specific gene of the invention). The inventioncovers any and all modified genes having the above characteristicswithout regard to the cause or means of modification or mutation.

The artificial means for said modification (mutation) of the amino acidsequence includes genetic engineering techniques such as site-specificmutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987); ibid.,100, 468 (1983); Nucleic Acids Res., 12, 9441 (1984); “Zoku SeikagakuJikken Koza (Experiments in Biochemistry, Second Series) 1”: IdenshiKenkyuho (Methods in Gene Research) II, the Biochemical Society of Japan(ed.), p 105 (1986), etc.], methods of chemical synthesis such as thephosphotriester method and phosphoamidite method [J. Am. Chem. Soc., 89,4801 (1967); ibid., 91, 3350 (1969); Science, 150, 178 (1968);Tetrahedron Lett., 22, 1859 (1981); ibid., 24, 245 (1983)], andcombinations of such methods.

More particularly, the DNA can be synthesized by a chemical method suchas the phosphoamidite method or the phosphotriester method, and thissynthesis can be effected on a commercially available automatedoligonucleotide synthesizer. The double-stranded fragment can beobtained from the chemically synthesized single-strand fragment bysynthesizing a complementary strand and annealing them under suitableconditions or adding the complementary strand using a suitable primersequence and a DNA polymerase.

A specific example of the gene according to the invention is the genehaving the nucleotide sequence shown in SEQ ID NO:3. The coding region(the sequence shown in SEQ ID NO:2) of this nucleotide sequence is anexample of combination of codons specifying the respective amino acidresidues of the amino acid sequence shown under SEQ ID NO:1. The gene ofthe invention is not limited to the gene having said defined nucleotidesequence but includes any gene having a nucleotide sequence obtainableby selecting any arbitrary combination of codons for each amino acidresidue. Selection of codons can be made in the routine manner, withreference to the codon usage in the host to be employed [Nucleic AcidsRes., 9, 43 (1981)].

Furthermore, the gene of the invention includes one having a nucleotidesequence showing a certain level of homology to the nucleotide sequenceshown in SEQ ID NO:3. Inferred by said level of homology arepolynucleotides and complementary polynucleotides having at least 70%homology, preferably at least 90% homology, more preferably at least 95%homology, to the nucleotide sequence shown in SEQ ID NO:3. The genehaving such a level of homology may for example be characterized as apolynucleotide which hybridizes with a DNA having the nucleotidesequence shown in SEQ ID NO:3 under stringent conditions. Moreparticularly, the gene having a nucleotide sequence which hybridizeswith the DNA having the nucleotide sequence shown in SEQ ID NO:3 underthe condition of 6×SSC at 65° C. overnight or 50% formamide-4×SSC at 37°C. overnight is subsumed in the concept of the gene having said level ofhomology. Here, SSC stands for standard saline citrate (1×SSC=0.15 MNaCl, 0.015 M sodium citrate).

The gene of the invention can be easily produced and isolated by thegeneral genetic engineering technology based on the sequence informationon any specific example of the gene of the invention as disclosed inthis specification [e.g. Molecular Cloning 2d Ed, Cold Spring HarborLab. Press (1989); Zoku Seikagaku Jikken Koza (Experiments inBiochemistry, Second Series): “Idenshi Kenkyuho (Methods in GeneResearch) I, II, III, the Biochemical Society of Japan (ed.), (1986)].

More particularly, this can be done by preparing a cDNA library from asuitable source, in which the gene of the invention can be expressed, bya routine procedure and selecting a desired clone from this libraryusing a suitable probe or antibody specific to the gene of the invention[Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 222, 778(1983)].

The source of cDNA, which can be used in the above procedure includesvarious cells and tissues expressing the gene of the invention, as wellas cultured cells derived therefrom, particularly brain tissues.Isolation of the total RNA from such a source, isolation andpurification of mRNA, and acquisition and cloning of cDNA can also becarried out in the conventional manner. Moreover, cDNA libraries arecommercially available and the present invention can be carried intopractice using such cDNA libraries, for example those cDNA librariesavailable from CLONTECH Lab. Inc.

The method of screening for the gene of the invention from a cDNAlibrary is not particularly restricted but the conventional procedurecan be employed. Examples of the screening methods include animmuno-screening method using a specific antibody to the proteinproduced by a cDNA to select the corresponding cDNA clone, a methodusing a probe selectively binding to the objective DNA sequence, such asa plaque hybridization method, and a colony hybridization method, and acombination of such methods.

As the probe for the above method, the DNA chemically synthesizedaccording to the nucleotide sequence information on the gene of theinvention can be generally employed. The gene of the invention which hasalready been obtained or a fragment thereof can also be used as theprobe with advantage. The sense primer and antisense primer establishedaccording to the nucleotide sequence information on the gene of theinvention can be used as screening probes.

The nucleotide sequence for use as the probe may be a partial nucleotidesequence corresponding to SEQ ID NO:2 and comprising at least 10consecutive nucleotides, preferably 20 consecutive nucleotides, morepreferably 30 consecutive nucleotides, most preferably 50 consecutivenucleotides. Moreover, the positive clone having the oligonucleotidesequence shown in SEQ ID NO:2 as such can be used as the probe.

In obtaining the gene of the invention, the DNA/RNA amplification by PCR[Science, 230, 1350 (1985)] can be used with advantage. Particularlywhen a full-length cDNA can hardly be obtained from a library, the RACEmethod [Rapid amplification of cDNA ends; Jikken Igaku (ExperimentalMedicine), 12(6), 35 (1994)], especially 5′-RACE method [M. A. Frohman,et al., Proc. Natl. Acad. Sci., USA., 8, 8998 (1988)], can be used withadvantage.

The primers for use in such PCR methods can be judiciously establishedwith reference to the sequence information on the gene of the inventionas disclosed herein and can be synthesized by the routine procedure. Theisolation and purification of the amplified DNA/RNA fragment can becarried out in the routine manner as mentioned above, for example by thegel electrophoresis method.

Sequencing of the gene of the invention as obtained in the above manneror various DNA fragments can be made in accordance with the dideoxymethod, [Proc. Natl. Acad. Sci., USA., 74, 5463 (1977)] or the Maxam andGilbert method [Methods in Enzymology, 65, 499 (1980)] or moreexpediently by using a commercial sequencing kit.

For example, with the gene of the invention thus obtained, theexpression or non-expression of the gene of the invention in anindividual or a given tissue can be specifically detected by utilizing aportion or the whole of the nucleotide sequence of the gene of theinvention.

The above detection can be made by the conventional procedures, such asRNA amplification by RT-PCR [reverse transcribed-polymerase chainreaction; E. S. Kawasaki, et al., Amplification of RNA. In PCR Protocol.A Guide to Methods and Applications, Academic Press, Inc., San Diego,21-27 (1991)], Northern blot analysis [Molecular Cloning, Cold SpringHarbor Lab. (1989)], determination on cellular level by in situ RT-PCR[Nucl. Acids Res., 21, 3159-3166 (1993)] or in situ hybridization, NASBA[nucleic acid sequence-based amplification, Nature, 350, 91-92 (1991)],and the like conventional techniques. The preferred is the RT-PCRdetection method.

The primer which is to be used when the PCR method is chosen for theabove purpose is not particularly restricted insofar as it ischaracteristic of the gene of the invention and capable of selectiveamplification of the particular gene only and can be judiciouslyestablished based on the sequence information on the gene of theinvention. Usually, one having a partial sequence of the gene of theinvention, which is about 10-35 nucleotides long, preferably about 15-30nucleotides long can be used as the primer.

The gene of the invention, thus, includes the DNA fragment which can beused as a specific primer and/or specific probe for the detection of theLY6H gene of the invention.

The DNA fragment mentioned above can be defined as a polynucleotidewhich hybridizes with the polynucleotide having the nucleotide sequenceshown in SEQ ID NO:2 under stringent conditions. The stringentconditions mentioned above may be the ordinary conditions for primers orprobes and, as such, are not particularly restricted. For example, theabove-mentioned conditions of 6×SSC, 65° C., overnight or the conditionof 50% formamide-4×SSC, 37° C., overnight can be mentioned.

By applying the gene of the invention to the standard geneticengineering technology, the expression product (polypeptide) of the geneor a protein containing it can be easily produced in large quantitiesand with good reproducibility.

Therefore, the invention further provides a polypeptide having the aminoacid sequence encoded by the gene of the invention (the expressionproduct of the invention), a vector harboring the gene of the inventionfor the production of the polypeptide, a host cell transfected with thevector, and a method of producing the polypeptide of the invention whichcomprises growing the host cell.

The polypeptide (LY6H protein) having the amino acid sequence shownunder SEQ ID NO:1 is a specific embodiment of the polypeptide of theinvention. The polypeptide of the invention is not limited to this LY6Hprotein but includes its homolog. The homolog may be a polypeptidehaving an amino acid sequence derived from the amino acid sequence shownin SEQ ID NO:1 by the deletion, substitution or addition of one or moreamino acids and retaining the same function as the LY6H protein. Aspecific example of the homolog is the expression product of a homologof said LY6H gene (the LY6H equivalent gene inclusive of the allele).

Furthermore, the homolog of the LY6H protein of the invention includesproteins having the same activity or function as the polypeptide havingthe amino acid sequence shown in SEQ ID NO:1 as derived from any ofmammals such as equine, sheep, bovine, canine, monkey, cat, bear, etc.and rodents such as rat, mouse and rabbit.

The polypeptide of the invention can be produced by the conventionalrecombinant DNA technology [e.g. Science, 224, 1431 (1984); Biochem.Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80,5990 (1983)]. based on the gene sequence information provided by thepresent invention.

More particularly, the production of said polypeptide is carried out bythe procedure comprising constructing a recombinant DNA (expressionvector) which permits expression of the gene coding for the desiredprotein in a host cell, transforming the host cell with the vector,growing the resulting transformant, and harvesting the polypeptide fromthe culture broth.

The host cell mentioned above may be whichever of a prokaryotic cell andan eukaryotic cell. As the prokaryotic host, Escherichia coli, Bacillussubtilits and other common bacteria can be mentioned and preferablycells of Escherichia coli, particularly cells of Escherichia coli K12,can be employed. The eukaryotic host cell includes cells of vertebratesand yeasts and the former include the monkey cell line COS [Cell, 23:175 (1981)], Chinese hamster ovarian cells, and the dihydrofolatereductase-defective cells thereof [Proc. Natl. Acad. Sci., USA., 77:4216 (1980)]. As the latter, yeast cells of the genus Saccharomyces canbe used with advantage, but these are not exclusive choices.

When prokaryotic cells are used as host cells, an expression plasmidconstruct prepared by using a vector which is replicatable in theparticular host cell and adding a promoter and SD (Shine and Dalgarno)sequence upstream of the gene of the invention so that the gene may beexpressed therein as well as an initiation codon (e.g. ATG) necessaryfor initiation of protein synthesis can be used with advantage. As thevector mentioned above, it is usual to employ plasmids derived fromEscherichia coli, such as pBR322, pBR325, pUC12, pUC13, etc. However,these are not exclusive choices but various known vectors can beutilized. Examples of the commercial vectors for use in expressionsystems using E. coli include pGEX-4T (Amersham Pharmacia Biotech),pMAL-C2, pMA1-P2 (New England Biolabs), pET21, pET21/lacq (Invitrogen)and pBAD/His (Invitrogen).

As the expression vector for use when cells of a vertebrate are used ashost cells, the vector having a promoter upstream of the gene of theinvention to be expressed, RNA splice sites, polyadenylation site and atranscription termination sequence is usually employed, and this vectormay further have a replication origin where necessary. A specificexample of the expression vector is pSV2dhfr harboring an early promoterof SV40 [Mol. Cell. Biol., 1: 854 (1981)]. Aside from the above, variousknown vectors available commercially can be employed. Examples of thecommercial vectors which are used in expression systems using animalcells include vectors for animal cells, such as pEGFP-N, pEGFP-C(CLONTECH), pIND (Invitrogen), pcDNA3.1/His (Invitrogen), etc., andvectors for insect cells, such as pFastBac HT (Gibci BRL), pAcGHLT(PharMingen), pAc5/V5-His, pMT/V5-His and pMT/Bip/V5-his (allInvitrogen).

pAM82 having a promoter for the acid phosphatase gene [Proc. Natl. Acad.Sci., USA., 80: 1 (1983)] is a specific example of the expression vectorfor use when yeast cells are used as host cells. The commercialexpression vectors for yeast cells include pPICZ (invitrogen) and pPICZα(Invitrogen).

The promoter is not particularly restricted, either. When a strain ofthe genus Escherichia is used as the host, tryptophan (trp) promoter,lpp promoter, lac promoter, recA promoter, PL/PR promoter, etc. can beutilized with advantage. When the host is a strain of the genusBacillus, SP01 promoter, SP02 promoter, penP promoter, etc. arepreferably used. When a yeast is used as the host, pH05 promoter, PGKpromoter, GAP promoter, ADH promoter, etc. can be utilized withadvantage. The preferred promoter for use when host cells are animalcells include SV40-derived promoters, retrovirus promoters,metallothionein promoter, heat shock promoter, cytomegalovirus promoter,and SRα promoter.

As the expression vector for the gene of the invention, the conventionalfusion protein expression vector can be used with advantage. pGEX(Promega) for the expression of glutathione-S-transferase (GST)-fusedproteins is a specific-example of the vector.

The polynucleotide sequence wherein the coding sequence for a maturepolypeptide assists in the expression and secretion of a polypeptidefrom host cells includes the secretory sequence, the leader sequence andthe marker sequence (hexahistidine tag, histidin tag) used in thepurification of a fusion mature polypeptide in the case of bacterialcells, and the hemaglutinin (HA) tag in the case of mammalian cells.

The method of introducing the recombinant DNA (expression vector) intothe host cell and the associated transforming method are notparticularly restricted but various standardized methods can beutilized.

The transformant obtained can be cultured in the routine manner, wherebythe objective protein encoded by the deliberately designed geneaccording to the invention is expressed and produced(accumulated/secreted) intracellularly, extracellularly or on the cellmembrane.

The culture medium to be used can be judiciously selected from amongvarious routine media according to the kind of adopted host cell and theculture is also performed under conditions favoring growth of the hostcell.

The resulting recombinant protein (LY6H protein) according to theinvention can be optionally isolated and purified by various separationtechniques taking advantage of its physical and/or chemical properties,for instance [“Seikagaku Data Book (Biochemical Data Book) II”,1175-1259, First Edition, 1st impression, Jun. 23, 1980, Tokyo KagakuDojin K.K.; Biochemistry, 25(25), 8274 (1986); Eur. J. Biochem., 163,313 (1987), etc.].

Examples of such techniques are the conventional reconstitution method,treatment with a protein precipitating agent (salting-out method),centrifugation, osmotic shock method, sonic disruption, ultrafiltration,various types of chromatography such as molecular sieve chromatography(gel filtration), adsorption chromatography, ion exchangechromatography, affinity chromatography and high performance liquidchromatography (HPLC), dialysis, and combinations of these techniques.The particularly preferred technique includes affinity chromatographyusing a column to which a specific antibody to the protein of theinvention has been coupled.

In designing the objective gene encoding the polypeptide of theinvention, the nucleotide sequence of Y6H gene as shown in SEQ ID NO:2can be utilized with advantage. If desired, this gene can be used afterthe codons specifying the respective amino acid residues have beenjudiciously altered. Furthermore, when any amino acid residue or partialsequence of the amino acid sequence encoded by the LY6H gene is to bemodified by substitution, deletion or addition, such modifications canbe made by the various methods described above, for example bysite-specific mutagenesis.

The polypeptide of the invention can be produced by the standardprotocol for chemical synthesis according to the amino acid sequenceshown in SEQ ID NO:1. The method includes the conventional liquid-phasemethod and solid-phase method for peptide synthesis.

More particularly, the method for peptide synthesis includes theso-called stepwise elongation method in which the constituent aminoacids are coupled one by one for chain extension and the fragmentcondensation method which comprises synthesizing fragments eachconsisting of several amino acids beforehand and coupling the fragmentstogether. The synthesis of the protein of the invention can be carriedout by whichever of the above two methods.

The method of condensation for use in the above peptide synthesis mayalso be a conventional one, including the azide process, mixed acidanhydride process, DCC process, active ester process, redox process,DPPA (diphenylphosphoryl azide) process, DCC+additive(1-hydroxybenzotriazole, N-hydroxysuccinamide,N-hydroxy-5-norbornene-2,3-dicarboximide or the like) process andWoodward's reagent process.

The solvent to be used in these processes can also be judiciouslyselected from among the common solvents well known in the art for use insuch peptide-forming condensation reactions. Examples of the solventsinclude dimethylformamide (DMF), dimethyl sulfoxide (DMSO),hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate, etc.,and mixtures thereof.

In conducting the peptide synthesizing reactions, the carboxyl group ofany amino acid or fragment peptide that should not take part in thereaction can be protected in advance, generally by esterification in theform of a lower alkyl ester such as methyl ester, ethyl ester,tert-butyl ester, etc. or an aralkyl ester such as benzyl ester,p-methoxybenzyl ester, p-nitrobenzyl ester, etc.

Referring to any amino acid having a functional group in its side chain,the hydroxyl group of a tyrosine residue, for instance, may be protectedin advance with an acetyl, benzyl, benzyloxycarbonyl, tertiary butyl orother group, although such protection is not necessarily indispensable.Furthermore, the guanidino group of an arginine residue can be protectedwith a suitable protective group such as nitro, tosyl,p-methoxybenzene-sulfonyl, methylene-2-sulfonyl, benzyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarboxyl or the like.

The reactions for eliminating such protective groups from the protectedamino acids, peptides or the end product protein of the invention canalso be carried out in the routine manner, for example by catalyticreduction or a method using liquid ammonia/sodium, hydrogen fluoride,hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid,formic acid, methane-sulfonic acid or other reagent.

The polypeptide of the invention, thus produced, can be purified asneeded by the various techniques mentioned above, such as ion exchangeresin chromatography, partition chromatography, gel chromatography,countercurrent distribution and the like methods in routine us in thefield of peptide chemistry.

The polypeptide of the invention can be used with advantage as animmunogen for preparation of its specific antibody. By utilizing thisimmunogen, the antiserum (polyclonal antibody) and the monoclonalantibody can be provided.

The technology of producing antibodies is well known to those skilled inthe art and the known procedures can be employed in the presentinvention [e.g. Zoku Seikagaku Jikken Koza (Experiments in Biochemistry,second series) “Men-eki Seikagaku Kenkyuho (Methods inImmunobiochemistry)”, edited by the Biochemical Society of Japan(1986)].

For example, as the immune animal for harvesting the desired antiserumtherefrom, the ordinary animals such as rabbit, guinea pig, rat, mouse,chicken, etc. can be arbitrarily selected and the immunization with saidimmunogen and the collection of blood can also be carried out by theconventional procedures.

Preparation of a monoclonal antibody can also be carried out by theconventional technique which comprises constructing a hybridoma betweenthe plasma cell (immune cell) of an animal immunized with said immunogenand a plasmacytoma cell, selecting clones producing the desiredantibody, and cultivating the clones. The immune animal is generallyselected in consideration of its compatibility with the plasmacytomacell to be used for cell fusion and usually the mouse or the rat is usedwith advantage. The immunization procedure may be the same as used forthe preparation of said antiserum and, if desired, the immunization canbe made using a conventional adjuvant in combination.

The plasmacytoma cell for use in said hybridization is not particularlyrestricted, either, but includes various myeloma cells such as p3(p3/x63-Ag8) [Nature, 256: 495-497 (1975)], p3-U1 [Current Topics inMicrobiology and Immunology, 81: 1-7 (1978)], NS-1 [Eur. J. Immunol., 6:511-519 (1976)], MPC-11 [Cell, 8: 405-415 (1976)], SP2/0 [Nature, 276:269-271 (1978)], etc., R210 [Nature, 277: 131-133 (1979)] and others inrats, and cells derived therefrom.

The hybridization between said immune cell and said plasmacytoma cellcan be effected by the known technology in the presence of aconventional hybridization promoter such as polyethylene glycol (PEG) orSendai virus (HVJ) and the separation of the objective hybridoma canalso be carried out in the known manner [Meth. in Enzymol., 73: 3(1981); Zoku Seikagaku Jikken Koza (ditto)].

The search for the objective antibody-producing cell clone and themonoclonal antibody preparation can also be carried out in the routinemanner. For example, the search for the antibody-producing hybridoma canbe made by any of the various techniques in routine use for thedetection of antibodies, such as ELISA [Meth. in Enzymol., 70: 419-439(1980)], plaque method, spot method, agglutination reaction method,Ouchterlony method, radioimmunoassay, and the like, using the protein ofthe invention as an antigen.

Harvesting of the antibody of the invention from the resulting hybridomacan be achieved by cultivating the hybridoma in the routine manner andrecovering the antibody as a culture supernatant or administering thehybridoma to a compatible mammal and recovering the antibody in the formof ascites. The former method is suitable for production of the antibodyof high purity, while the latter method is suitable for high-productionof the antibody. The antibody thus produced can be further purified bythe conventional means such as salting-out, gel filtration, affinitychromatography and the like.

The antibody thus obtained is characterized by its binding affinity forthe LY6H protein of the invention and can be used with advantage for thepurification of LY6H protein and determination or differentiation of theprotein by immunological techniques. Furthermore, since a decreasedexpression of the gene of the invention has been confirmed in thetemporal lobe of the brain of a patient with Alzheimer's disease whichis a neurodegenerative disease, this antibody can be utilized in thescreening for agonists or antagonists of LY6H protein.

The present invention provides the novel antibody described above, too.

The polypeptide of the invention is useful in the field of medicine aspharmaceutical products containing it as an active ingredient.Therefore, the invention provides a pharmaceutical compositioncomprising the polypeptide of the invention as an active ingredient.

The usefulness of the polypeptide of the invention in or as saidpharmaceutical composition is ascribable to the neuronalsurvival-supporting action, nerve elongating action, nerve regeneratingaction, neuroglia-activating action and mnemonic action inherent in thisbrain-specific polypeptide. Examples of the methods for confirming theseactions include the following methods for each action.

1) Neuronal Survival-supporting Action

The following method can be used for quantitating the neuronalsurvival-supporting action of the polypeptide of the invention. Forexample, the hippocampus is aseptically isolated from the whole brain ofa fetal SD rat and treated with an enzyme, and seeded in a poly-L-lysine(Sigma)-precoated 96-well plate containing 10% fetal calf serum-DMEM ata final concentration of 2×10⁵ cells/cm².

The cells are grown for 24 hours, at the end of which time the culturemedium is changed to 1% N2 Supplement (Gibco)-containing DMEM. Then, theactive ingredient polypeptide of the invention is added (the inventiongroup). As control, the polypeptide of the invention which has beenheat-treated in a boiling water bath for 5 minutes is added (the boiledprotein group).

The cells (culture) in each group as prepared in the above manner arecultured for 72 hours. Then, by performing an MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assayusing Promega Cell Titer 96-Well Assay System, the neuronalsurvival-supporting effect of the polypeptide of the invention onhippocampal neurons can be evaluated.

Similarly, by isolating the ventral midbrain aseptically from the samewhole brain of a fetal SD rat as above and carrying out an MTT assay inthe same manner as above, the neuronal survival-supporting effect of thepolypeptide of the invention on the midbrain neurons can beinvestigated.

2) Dopaminergic Neuron Survival-supporting Action

As a method of evaluating the neuronal survival-supporting action of thepolypeptide of the invention, the following method of quantitating thedopaminergic neuron survival-supporting activity can be mentioned. Thus,the cells (culture) in each group as prepared above under 1) arecultured for 72 hours and, then, fixed with 4% paraformaldehyde-PBS by15-minute standing at room temperature. Then, using 1% Triton×100/PBS,the culture is passed through a membrane.

To prevent non-specific binding of the antibody, the cells are incubatedin 10% goat serum-PBS for 1 hour and, then, using an anti-tyrosinehydroxylase polyclonal antibody (Chemicon, diluted 1000-fold in PBS),further incubated at 4° C. for 16 hours. After removal of the antibodyfluid, the cells are washed with PBS and, after addition ofperoxidase-labeled dextran polymer-coupled goat anti-rabbitimmunoglobulin (Dako), are incubated at room temperature for 1 hour.

Detection of the tyrosine-hydroxylase-positive cells can be made by thecolor reaction using diaminobenzidine as the substrate. In this manner,the dopaminergic neuronal survival-supporting activity of thepolypeptide of the invention can be assayed using the number of tyrosinehydroxylase-positive cells as the indicator.

3) Nerve Elongating Action

The determination of the nerve elongating action (axonalelongation-promoting action) of the polypeptide of the invention can becarried out using PC12 cells [ATCC Accession Number CRL1721; Science,229, 393-395 (1985)] as follows. Thus, PC12 cells subcultured inmodified Dalbecco's MEM (D-MEM) containing 5% of heat-inactivated (56°C., 30 min) horse serum and 10% fetal calf serum (FCS) are transplantedin a collagen-coated plastic petri dish, 35 mm in diameter, at aconcentration of 6×10⁴ cells/3 ml. On day 2 after transplantation, themedium was replaced with D-MEM containing a varying concentration of thepolypeptide of the invention as well as nerve growth factor (NGF; WakoPure Chemical Ind.) and FCS and the cultivation is continued in eachcase. On day 3, morphological changes of the cells are examined with aphase-contrast microscope. By assessing whether the formation ofneurites or the promotion of neurite outgrowth is observed in comparisonwith control, the axonal elongation-promoting potential of thepolypeptide of the invention can be evaluated.

4) Neuroglia-activating Action

The neuroglia-activating action can be evaluated, for example bydetermining the effect of the polypeptide of the invention on theactivation of neuroglia by FGF in accordance with the method of Kniss etal. or the method of Bogler et al. [Kniss, D. A., and Burry, R. W.,Brain Res., 439, 281-288 (1988); Bogler, O., et al., Proc. Natl. Acad.Sci., USA., 87(16), 6368-6372 (1990)].

5) Mnemonic Action

The mnemonic action can be evaluated, for example, in accordance withthe water-maze protocol of Morris [Morris. R. G. M., J. Neurosci. Meth.,11, 47-60 (1984)].

Another evaluation method comprises administering the LY6H protein or anagonist or antagonist of the LY6H protein as selected by a screening toan animal model of Alzheimer's disease such as a mutant β-amyloidprecursor protein gene or mutant presenilin 1 gene transgenic mouse[e.g. Nature, 373, 523-527 (1995); Nature Med., 5, 560-564 (1999)] andevaluating the degree of progression of the disease or the degree ofnerve degeneration in comparison with a non-treated control group.

Moreover, in order to have the gene expressed in the human temporal lobe(gene therapy), an adenovirus vector [Straus, E. S., Plenum Press NewYork, 451-496 (1984); Setoguchi, Y., et al., Blood, 84, 2953-2964(1994)], for instance, is used. Thus, a possible procedure comprisescloning the gene of the invention in an adenovirus vector, culturing itin the stem cell, administering it directly into the temporal lobe orintravenously through a peripheral blood vessel and checking to seewhether Alzheimer type dementia or Alzheimer's disease has been improvedor its progression inhibited.

The polypeptide as the active ingredient of the pharmaceuticalcomposition of the invention includes its pharmaceutically acceptablesalt. Such salt includes nontoxic salts with alkali metals, alkalineearth metals or ammonium, such as salts with sodium, potassium, lithium,calcium, magnesium, barium and ammonium. These salts can be prepared bythe conventional methods in the art. Furthermore, said salt includesnontoxic acid addition salts which can be prepared by reacting theactive ingredient polypeptide of the invention with suitable organic orinorganic acids. The representative nontoxic acid addition salt includesthe hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate,valerate, oleate, laurate, borate, benzoate, lactate, phosphate,p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate,tartrate, sulfonate, glycolate, maleate, ascorbate, benzenesulfonate,naphthalenesulfonate, and the like.

The pharmaceutical composition of the invention includes a compositioncomprising a pharmacologically effective amount of the polypeptide ofthe invention and a suitable nontoxic pharmaceutical carrier or diluent.

The pharmaceutical carrier which can be used for said pharmaceuticalcomposition (pharmaceutical preparation) includes diluents or excipientswhich are conventionally utilized according to dosage forms, such asfillers, volume builders, binders, humectants, disintegrators,surfactants, and lubricants. These can be judiciously selected and usedaccording to the unit dosage form of the composition.

The particularly preferred pharmaceutical composition of the inventioncan be prepared using various additives which can be formulated inordinary protein preparations, such as the stabilizer, biocide, buffer,isotonizing agent, chelating agent, pH control agent and surfactant.

The stabilizer includes human serum albumin, an L-amino acid, a sugar,and a cellulose derivative, for instance, can be mentioned. These may beused singly or in combination with a surfactant or the like wherenecessary. The use in combination with a surfactant may lead to a moreeffective stabilization of the active ingredient in particular.

The L-amino acid is not particularly restricted but may for example beany of glycine, cysteine and glutamic acid.

The sugar is. not particularly restricted but includes monosaccharidessuch as glucose, mannose, galactose, fructose, etc.; sugar alcohols suchas mannitol, inositol, xylitol, etc., disaccharides such as sucrose,maltose, lactose, etc.; polysaccharides such as dextran,hydroxypropylstarch, chondroitin sulfate, hyaluronic acid, etc.; andtheir derivatives.

The surfactant is not particularly restricted, either, but both ionicand nonionic surfactants can be employed. Examples of the surfactant arepolyoxyethylene glycol sorbitan alkyl esters, polyoxyethylene alkylethers, sorbitan monoacyl esters and fatty acid glycerides.

The cellulose derivative that can be used is not particularlyrestricted, either, but includes methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, and carboxymethylcellulose sodium.

The level of addition of any of said sugar and other additives can bejudiciously selected with reference to the amount in common use.Generally, the sugar is used in a proportion of not less than about0.0001 mg, preferably within the range of about 0.01 to about 10 mg, perμg of the active ingredient. The surfactant is used generally in aproportion of not less than about 0.00001 mg, preferably within therange of about 0.0001 to about 0.01 mg, per μg of the active ingredient.Human serum albumin, an example of the stabilizer, can be used in aproportion of not less than about 0.0001 mg, preferably within the rangeof about 0.001 to about 0.1 mg, per μg of the active ingredient. Theamount of the amino acid, another example of the stabilizer, can beselected from the range of about 0.001 to about 10 mg per μg of theactive ingredient. The level of addition of the cellulose derivative isnot less than about 0.00001 mg and is preferably selected from the rangeof about 0.001 to about 0.1 mg.

The amount of the active ingredient in the pharmaceutical composition ofthe invention can be liberally selected from a broad range but isgenerally selected from the range of about 0.00001 to about 70 weight %,preferably about 0.0001 to about 5 weight %.

The pharmaceutical composition of the invention may be furthersupplemented with a buffer, an isotonizing agent, and a chelating agent.The buffer includes boric acid, phosphoric acid, acetic acid, citricacid, ε-aminocaproic acid, glutamic acid, and the corresponding salts(the alkali metal or alkaline earth metal salts thereof, such as sodiumsalts, potassium salts, calcium salts and magnesium salts). Theisotonizing agent includes sodium chloride, potassium chloride, sugarsand glycerol. The chelating agent includes sodium edetate, and citricacid. The level of addition of any of these additives may be within theconventional range.

The pharmaceutical preparation of the invention can be provided in theform of a solution, and in a lyophilized form which can be stored. Suchlyophilized preparations can be extemporaneously dissolved in, forexample, a buffer inclusive of water, saline or the like at a suitableconcentration.

As regards the unit dosage form of the pharmaceutical composition of theinvention, various forms can be selected according to the therapeuticobjective. The representative form includes solid dosage forms such astablets, pills, powders, neat powders, granules, capsules, etc. andliquid dosage forms such as solutions, suspensions, emulsions, syrups,elixirs and so on. These dosage forms are generally classified, by routeof administration, into oral preparations, parenteral preparations,nasal preparations, vaginal suppositories, rectal suppositories,sublingual tablets, ointments, and others. Each of such dosage forms canbe formulated and molded or otherwise prepared by the establishedpharmaceutical procedure.

For example, tablets can be manufactured using, as said pharmaceuticalcarrier, any of various excipients such as lactose, sucrose, sodiumchloride, glucose, urea, starch, calcium carbonate, kaolin, crystallinecellulose, silicic acid, potassium phosphate, etc.; binders such aswater, ethanol, propanol, simple syrup, glucose solution, starchsolution, gelatin solution, carboxymethylcellulose,hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone, etc.;disintegrators such as carboxymethylcellulose sodium,carboxymethylcellulose calcium, low-substitution-degreehydroxypropylcellulose, dried starch, sodium alginate, agar powder,laminaran powder, sodium hydrogen carbonate, calcium carbonate, etc.;surfactants such as polyoxyethylene sorbitan fatty acid esters, sodiumlauryl sulfate, monoglycerol stearate, etc.; disintegration inhibitorssuch as sucrose, stearin, cacao butter, hydrogenated oil, etc.;absorption promoters such as quaternary ammonium bases, sodium laurylsulfate, etc.; humectants such as glycerol, starch, etc.; adsorbentssuch as starch, lactose, kaolin, bentonite, colloidal silica, etc.; andlubricants such as purified talc, salts of stearic acid, boric acidpowder, polyethylene glycol and so on.

Where necessary, such tablets can be coated with conventional coatingsto provide sugar-coated tablets, gelatin-coated tablets, enteric-coatedtablets and film-coated tablets. Double-layer or multi-layer tablets mayalso be employed.

The pharmaceutical carrier which can be used for the production of pillsincludes various excipients such as glucose, lactose, starch, cacaobutter, hydrogenated vegetable oil, kaoline, talc, etc.; binders such asgum arabic powder, tragacanth powder, gelatin, ethanol, etc.; anddisintegrators such as laminaran and agar.

The capsules can be generally manufactured in the conventional manner byblending the active ingredient of the invention with the pharmaceuticalcarrier or carriers and filling the resulting composition into hardgelatin capsule shells, soft capsule shells or the like.

The liquid preparation for oral administration includes pharmaceuticallyacceptable solutions, emulsions, suspensions, syrups, elixirs, etc. asformulated with routine inert diluents, such as water, and these dosageforms may contain a wetting agent, an emulsifier, a suspending agentand/or other auxiliary additives. These can be manufactured by theestablished pharmaceutical procedures.

The liquid preparation for parenteral administration, inclusive ofsterile aqueous and non-aqueous solutions, emulsions and suspension, canbe prepared using such diluents as water, ethyl alcohol, propyleneglycol, polyethylene glycol, ethoxylated isostearyl alcohol,polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acidesters, and vegetable oils such as olive oil. In addition, injectableorganic esters, such as ethyl oleate, may be formulated. Furthermore,any of the conventional solubilizers, buffers, wetting agents,emulsifiers, suspending agents, preservatives, dispersants, etc. canalso be added.

The above various pharmaceutical dosage forms are sterilized in theroutine manner. This sterilization can be achieved by filtration througha bacterial filter, formulation of a biocide, irradiation, or a heattreatment. Furthermore, these may be provided in the form of sterilesolid compositions which can be extemporaneously dissolved in sterilewater or a suitable sterilizable medium.

For the manufacture of dosage forms for rectal or vaginaladministration, such pharmaceutical carriers as polyethylene glycol,cacao butter, higher alcohols, higher alcohol esters, gelatin,semisynthetic glycerides, etc. can be employed.

Ointments such as pastes, creams and gels can be prepared using adiluent such as white petrolatum, paraffin, glycerol, cellulosederivatives, propylene glycol, polyethylene glycol, silicone, bentonite,and vegetable oils such as olive oil.

Compositions for transnasal or sublingual administration can be preparedin the routine manner using a well-known standard excipient.

Where necessary, those pharmaceutical preparations of the invention maybe supplemented with coloring agents, preservatives, perfumes, flavoringagents, sweeteners, and other drugs.

The method for administration of such pharmaceutical preparations is notparticularly restricted but can be selected according to the specificdosage form, patient's age, sex and other factors, severity of illness,and other variables. For example, tablets, pills, solutions,suspensions, emulsions, granules and capsules are administered orally,while parenteral products are administered intravenously, either aloneor in admixture with the conventional glucose, amino acid or otherinfusion, or, where necessary, administered alone intramuscularly,intradermally, subcutaneously or intraperitoneally. The rectalsuppositories are administered into the rectum; the vaginalsuppositories are administered into the vagina; the nasal preparationsare administered into the nostrils, the sublingual preparations areadministered buccally, and the ointments are administered topically fortransdermal drug delivery.

The dosage for any of the above pharmaceutical preparations is notparticularly restricted but can be judiciously selected from a broadrange according to the expected therapeutic effect, administrationmethod, duration of treatment, patient background such as age and sex,and other factors. Generally, the recommended usual dosage of the activeingredient is about 0.01 μg-10 mg/day, preferably about 0.1 μg-1 mg/day,per kg of the patient's body weight. The above dose may be administeredonce a day or in 2 or more divided doses.

Furthermore, as pointed out in the working example to be presented laterherein, the expression of the gene of the invention has been abolishedor decreased in the temporal lobe of patients with Alzheimer's disease.Therefore, by constructing an arbitrary expression vector harboring thewhole or part of the gene of the invention and introducing theexpression vector into the temporal lobe tissue for forced expression ofthe gene in the tissue, neurodegenerative changes inclusive of anexcessive atrophy of neurons in the temporal lobe may be inhibited and,hence, the progression of Alzheimer's disease may be arrested.Therefore, the present invention further provides a pharmaceuticalcomposition for gene therapy (gene therapeutic agent) which is possessedof such a neurodegeneration-inhibitory action.

The present invention further provides the above expression vector orvector for gene therapy, cells transfected with the gene of theinvention through the introduction of said vector, and a pharmaceuticalcomposition for gene therapy which comprises any of the above as theactive component.

The gene therapy using said gene therapeutic agent is performed byadministering at least one member selected from the group consisting ofthe vector for introduction and expression of the gene of the inventionand cells transfected with the gene of the invention through theintroduction of said vector into the brain neurons or temporal lobetissue of a patient with neurodegenerative disease. By such a procedure,neurodegenerative changes in such tissue can be inhibited and symptomsof Alzheimer's disease, Alzheimer type dementia, Parkinson's disease,brain ischemia, etc. can be alleviated.

The gene therapy is now described in further detail. In the followingexecution of a gene therapy, the routine chemical, molecular biological,microbiological, recombinant DNA, genetic, and immunological techniquescan be employed unless otherwise specified. These techniques aredescribed in, inter alia, Maniatis, T., et al., Molecular Cloning: Alaboratory manual (Cold Spring Harbor Laboratory), Cold Spring Harbor,N.Y. (1982)), Sambrook, J., et al., Molecular Cloning: A laboratorymanual, 2nd Ed. (Cold Spring harbor Laboratory), Cold Spring harbor,N.Y. (1981)), Ausbel, F. M., et al., Current Protocols in MolecularBiology, John Wiley and Sons, New York (1992)), Glover, D., DNA Cloning,I and II (Oxford Press (1985)), Anand, Techniques for the Analysis ofComplx Genomes (Academic Press (1992), Guthrie, G., et al., Guide toYeast Genetics and Molecular Biology (Academic Press (1991)), and Fink,et al., Hum. Gene Ther., 3, 11-19 (1992).

The gene therapy can be carried out using a gene therapy vectorharboring the whole or part of the gene of the invention or cellstransfected with the gene of the invention through the introduction ofsaid vector. This gene therapy may for example be a method of supplyingthe LY6H gene or its function to cells in which said gene has not beenexpressed. By such gene therapy, neurodegeneration around the receptorcell/target cell is inhibited.

The gene of the invention or a fragment of the gene can be introducedinto cells by means of a vector adapted to maintain the geneextrachromosomally. In such cases, the particular gene can be caused tobe expressed by the cells from an extrachromosomal position. Moreover,when the LY6H gene is to be expressed by introducing a fragment of thegene into the temporal lobe site of the brain nervous system where noexpression of the gene is found, the particular fragment of the gene maybe a fragment encoding a part of LY6H protein which is necessary for thesurvival or non-tumorigenic growth of cells.

The gene transfer vector may be any of various known vectors in whichthe gene of the invention has been subcloned as will be described laterherein.

The introduction of the gene transfer vector into the target cell can beeasily effected by the established technology of introducing DNA intovarious cells which is already known to those skilled in the art, suchas electroporation, calcium phosphate transfection (coprecipitation),virus transduction and other techniques. The cells transfected with thegene of the invention can be utilized as a drug for neurodegenerativedisorders of the brain, inclusive of an inhibitor of premature atrophyof the brain nervous system, or as models for therapeutic research.

As mentioned above, the gene or gene fragment of the invention asintroduced by the gene therapy according to the invention increases theexpression of the corresponding gene product in the brain nerve orsurrounding tissue to thereby inhibit atrophy of the brain nerve in thetissue expressing the gene. Such gene therapy can be applied withadvantage to the brain neuronal tissue where the expression of LY6H geneor the LY6H protein has been abolished as well as to the brain neuronaltissue where the level of expression of said gene has been depressed.

The gene therapy according to the present invention is performed asfollows. First, a screening is carried out for candidate patients forthe gene therapy by recording a computer tomogram (CT) with the scanningposition fixed to the temperal lobe of the patient with Alzheimer typedementia or Alzheimer's disease to check for atrophy of the temporallobe or progression of the atrophy.

Then, to achieve expression of the gene of the invention, theintracellular LY6HmRNA is created in the target cell and its translationis promoted to accelerate expression of the LY6H gene. For this purpose,preferably a sense oligonucleotide corresponding to the mRNA of the geneis produced and supplied to the target cell. By providing the cell withthe activity to promote expression of the LY6H gene by the above genetherapy, the neurodegenerative change in the brain receptor cell/targetcell can be inhibited.

According to the above gene therapy using said sense oligonucleotide,the objective inhibition of neurodegenerative change of the brain andconsequent alleviation or arrest of progression of neurodegenerativesymptoms may be successfully attained by subcloning the LY6H gene into aretrovirus, adenovirus or AAV-derived vector and infecting the targetbrain nerve cells with the vector to thereby cause expression of thesense oligonucleotide.

When a sense oligonucleotide of the gene of the invention is introducedinto the cerebral neuron or tissue to increase the expression of LY6Hprotein, the sense oligonucleotide need not be the full-lengthnucleotide of the LY6H gene but may be the modification product insofaras it retains a function substantially identical to the function of theparent gene and promotes expression of the LY6H gene or a fragment genecomprising a partial sequence retaining said function.

Vectors which can be used for introducing an objective gene for both DNArecombination and extrachromosomal gene maintenance are already known inthe art and any of such known vectors can be used in the practice of theinvention. For example, a virus or plasmid vector which includes a copyof LY6H gene sense oligonucleotide ligated to the expression controlelement and is capable of expressing the sense-oligonucleotide productin the target cell can be used as such vectors. Any of the expressionvectors mentioned above can be usually employed but the preferred arevectors constructed by using any of the vectors disclosed in USP 5252479and WO 93/07282 (specifically pWP-7A, pWP-19, pWU-1, pWP-8A, pWP-21and/or pRSVL) or the pRC/CMV (Invitrogen) as the source vector. Thestill more preferred are the various virus vectors described laterherein.

As the promoter for use in the vector for gene therapy, those promoters,which are intrinsic to the affected tissues to be treated in variousdiseases, are preferably employed. Examples of the promoters arealbumin, α-fetoprotein, α1-antitrypsin, transferrin, and transthyretinfor the liver, and carbonic anhydrase I and carcinoembryonic antigen forthe colon. When the affected tissues are the uterus and placenta,estrogen, aromatase, cytochrome P450, cholesterol side-chain-cleavingenzyme P450, and 17α-hydroxylase P450 can be exemplified.

For the prostate, prostate-specific antigens, gp91-fox gene, andprostate-specific kallikrein can be exemplified. For the breast, erb-B2,erb-B3, β-casein, β-lactoglobin, and whey protein can be exemplified.For the lung, surfactant protein C, and uroglobulin can be exemplified.For the skin, K-14-keratin, human keratin 1 or 6, and leucline can beexemplified. For the brain, glial fibrillary acidic protein, matureastrocyte-specific protein, myelin, tyrosine hydroxylase pancreaticvillin, glucagon, and Langerhans islet amyloid polypeptide can beexemplified. For the thyroid, thyroglobulin, and calcitonin can beexemplified. For the bone, α1 collagen, osteocalcin, and bonesialoglycoprotein can be exemplified. For the kidney, renin,liver/bone/kidney alkaline phosphatase, and erythropoietin can beexemplified, and for the pancreas, amylase, and PAP1 can be exemplified.

Furthermore, in the production of a vector for introduction of a senseoligonucleotide, the sense oligonucleotide to be introduced (one havinga full-length or partial sequence corresponding to the sequence of thegene of the invention) can be easily prepared and acquired by thestandard genetic engineering techniques based on the nucleotide sequenceinformation on the gene of the invention as described hereinbefore.

The transfer of such a vector for introduction of a senseoligonucleotide into cells can be carried out by various techniquesalready known in the art, such as electroporation, calcium phosphatetransfection (coprecipitation), virus transduction and the like. Thecells transfected with said sense oligonucleotide, as such and in anisolated form, have a brain neurodegeneration-inhibitory action so thatthey can be used as a drug, or a therapeutic research model, for theinhibition or arrest of progression of neurodegenerative lesions aswell.

In gene therapy, the above vector for introduction of a senseoligonucleotide can be injected either topically into the temporal lobeor surrounding region of the patient or systemically. Furthermore, itmay be cultured together with stem cells and, then, administered bylocal or systemic injection. By such administration, the vector can beintroduced into the nerve cells of the patient's brain. In the event thetransduced gene is not permanently taken up in the chromosome of eachtarget cell, the administration may be repeated periodically.

The method for gene therapy according to the invention includes both thein vivo technique which comprises administering a construct forintroduction of said sense oligonucleotide (a sense oligonucleotidetransfer vector) directly into the body and the ex vivo technique whichcomprises transferring the gene into cultured stem cells and, afterculturing, transplanting or otherwise introducing the cells into thepatient's body. A gene therapy comprising introducing said senseoligonucleotide directly into the cell is also feasible.

The target cells into which the sense oligonucleotide of the gene of theinvention is to be introduced can be judiciously selected according tothe object of gene therapy (treatment). For example, the target cellsinclude brain neurons and brain nerve tissues as well as lymphocytes,fibroblasts, hepatocytes and hemopoietic cells.

The method of introducing the sense oligonucleotide in the above genetherapy includes a viral introduction technique and a non-viralintroduction technique.

As to the viral introduction technique, in consideration of the factthat the sense oligonucleotide to be transferred is a foreign substancewhich is expressed especially in the normal brain cells, the methodusing a retrovirus vector, for instance, can be exemplified. Other virusvectors which can be used include the adenovirus vector, HIV (humanimmunodeficiency virus) vector, adeno-associated virus (AAV) vector,herpes virus vector, herpes simplex virus (HSV) vector, and Epstein-Barrvirus (EBV) vector.

The method of constructing a virus vector for transfer of a senseoligonucleotide and the method for transfer of the sense oligonucleotideto the target cell or target tissue are now specifically described.

The retrovirus vector system consists of a virus vector and a helpercell (packaging cell). The helper cell means a cell which has expressedgenes encoding the structural protein gag (structural protein within thevirus particle), pol (reverse transcriptase), env (coat protein), etc.of a retrovirus but which has not formed virus particles. On the otherhand, the virus vector has the packaging signal and LTR (long terminalrepeats) but lacks structural genes, such as gag, pol, env, etc., whichare necessary for virus replication. The packaging signal is a sequencewhich functions as a tag in the assembly of a virus particle. Selectivegenes (neo, hyg) and the object sense oligonucleotide ligated in thecloning site are inserted in lieu of the virus genes. In order that ahigh titer of virus particles may be obtained, it is important to use aninsert as short as possible, provide a broad packaging signal includinga part of the gag gene, and use care not to leave ATG of the gag gene.

As the vector DNA harboring the object sense oligonucleotide istransferred to the helper cell, the vector genomic RNA is packaged bythe virus structural protein formed by the helper cell, whereby virusparticles are formed and secreted. The virus particle as a recombinantvirus infects the target cell and, as a result, the DNA sequencereverse-transcribed from the virus genomic RNA is integrated into thecell nucleus so that the sense gene inserted in the vector is expressed.

It may be employed a technique using a fibronectin fragment containingthe cell adhesion domain, heparin-binding site and conjugating segment[Hanenberg, H., et al., Exp. Hemat., 23, 747 (1995)], for enhancing theefficiency of transfer of the object gene.

An example of the retrovirus vector for use in the above retrovirusvector system is the retrovirus derived from mouse leukemia virus[McLachlin, J. R., et al., Proc. Natl. Acad. Res. Molec. Biol., 38,91-135 (1990)].

The method using an adenovirus vector is now described in detail. Theadenovirus vector can be constructed in accordance with the methodsdescribed in Berkner, K. L., Curr. Topics Microbiol. Immunol., 158,39-66 (1992), Setoguchi, Y., et al., Blood, 84, 2946-2953 (1994),Kanegae, H. et al. [Jikken Igaku (Experimental Medicine), 12, 28-34(1994)] and Ketner, G. et al., Proc. Natl. Acad. Sci., USA., 91,6186-6190 (1994).

For example, to construct a non-proliferative adenovirus vector, theearly region E1 and/or E3 of the adenovirus is excised in the firstplace. Then, a plasmid vector containing the desired foreign geneexpression unit (which consists of the sense oligonucleotide to betransferred, the promoter for transcription of said senseoligonucleotide, Poly A for insuring the stability of the transcribedgene) and a part of the adenovirus genomic DNA and a plasmid containingthe adenovirus genome are used to cotransfect the 293 cell, forinstance. As a homologous recombination is thus caused to take placebetween them for substitution of the gene expression unit for E1, anonproliferative adenovirus vector is obtained as a vector harboring theobject sense oligonucleotide. A 3′-end adenovirus vector with a terminalprotein added can also be constructed by ligating the adenovirus genomicDNA in a cosmid vector. Furthermore, the YAC vector may also be utilizedfor the construction of an adenovirus vector.

Production of an adeno-associated virus (AAV) vector is now describedbriefly. AAV was discovered as a small virus contaminating adenovirusculture systems. As to this virus, the existence of the genus Parvoviruscapable of autonomous proliferation within the host cell withoutrequiring a helper virus for virus replicatioon and the genusDependovirus which requires a helper virus has been identified. This AAVhas a broad host range and is one of the common viruses infectingvarious kinds of cells. The virus genome is a linear single-stranded DNAconsisting of 4680 nucleotides, with the 145 nucleotides at both endshaving a characteristic sequence known as ITR (inverted terminalrepeat). This ITR region functions as the replication origin and playsthe role of a primer. This ITR is also essential to packaging for virusparticles and integration of AAV into the chromosome DNA of the hostcell. In regard of the virus protein, the left-half of the genome codesfor the nonstructural protein, that is the regulatory protein Rep whichcontrols replication and transcription.

Construction of the recombinant AAV can be carried out by utilizing theproperty of AAV to become integrated into the chromosome DNA, wherebythe desired gene transfer vector can be prepared. This method may bedescribed in detail as follows. First, a plasmid (AAV vector plasmid)retaining the ITRs at 5′- and 3′-ends of a wild-type AVV and harboringthe sense oligonucleotide to be transferred as interposed therebetweenis constructed. The virus protein necessary for virus replication andformation of virus particles is supplied from a separate helper plasmid.It is necessary to insure that no common nucleotide sequence will existbetween the two plasmids so that a wild-type virus will not appear onDNA recombination. Thereafter, the two plasmids are transferred into the293 cell by transfection, for example, and, further, the cells areinfected with an adenovirus as the helper virus (when the 293 cell isused, this adenovirus may be a non-proliferative one), whereby thedesired non-proliferative recombinant AAV is produced. Since thisrecombinant AAV is present in the nucleus, the cells are subjected tofreeze-thawing and recovered and the contaminant adenovirus isinactivated by heating at 56° C. Then, where necessary, the recombinantAAV is separated and concentrated by ultracentrifugation using cesiumchloride. In this manner, the desired recombinant AAV for gene transfercan be obtained.

Production of an EBV vector can be carried out by the method of Shimidzuet al. [Shimidzu, N., SAIBO KOUGAKU (Cell Technology, 14(3), 280-287(1995)].

Production of the EBV vector for transfer of the sense oligonucleotideaccording to the invention is now described briefly. EB virus(Epstein-Barr virus) is a virus of the family Herpesviridae, which wasfirst isolated by Epstein and coworkers from cultured cells derived fromBurkitt lymphoma [Kieff, E. and Liebowitz, D.: Virology, 2nd ed. RavenPress, New York, 1990, pp. 1889-1920]. This EBV has cell-transformingactivity and in order to use it as a vector for gene transfer, it isnecessary to prepare a virus defected of this transforming activity.This can be done as follows.

Thus, first of all, the EBV genome in the vicinity of the target DNA inwhich the desired foreign gene is to be inserted is cloned. Then, a DNAfragment of the foreign gene and a drug-resistant gene are inserted toconstruct a vector for preparation of a recombinant virus. Then, thevector for preparation of a recombinant virus as excized with a suitablerestriction enzyme is transfected to EBV-positive Akata cells. Therecombinant virus formed by homologous recombination is recovered,together with the wild type Akata EBV, through stimulation of virusproduction by anti-surface immunoglobulin treatment. The recombinantvirus is infected to EBV-negative Akata cells and, in the presence of adrug, resistant clones are selected, whereby Akata cells infectedexclusively with the recombinant virus free of wild type EBV can beobtained. Further, by inducing viral activity in the recombinantvirus-infected Akata cells, the objective recombinant virus vector canbe produced in quantities.

The method of introducing the object gene into the target cell or targettissue in the gene therapy of the invention includes the followingrepresentative two methods.

The first method comprises harvesting the target cells from a patient tobe treated, growing the cells ex vivo, for example under addition ofinterleukin-2 (IL-2) or the like, to transfer the objective senseoligonucleotide harbored in the retrovirus vector, and retransplantingthe resulting cells (ex vivo method). This method is suitable for thetherapy of genetic diseases caused by defective genes and cancer, forinstance.

The second method is a method for direct gene transfer which comprisesinjecting the object sense oligonucleotide directly into the patient'sbody or the target site such as the cerebral tissue (direct method).

More particularly, the first method can be carried out in the followingmanner, for instance. Thus, the mononuclear cells, such as stem cells,harvested from the patient are fractionally separated from monocytesusing a blood sorter and cultured in the presence of IL-2 in a suitablemedium such as AIM-V medium for about 72 hours, followed by addition ofthe vector harboring the sense oligonucleotide to be introduced. Forenhancing the efficiency of transfer of the sense oligonucleotide, thecells may be grown in the presence of protamine at 32° C. for 1 hour,centrifuged at 2500 ppm, and then cultured under 10% carbon dioxide gasat 37° C. for 24 hours. After this procedure is repeated a few times,the cells are further cultured in the presence of IL-2 in, for example,AIM-V medium for 48 hours and then washed with saline. The viable cellsare counted and the efficiency of introduction of the senseoligonucleotide is evaluated by said in situ PCR or, when the object isenzymatic activity, assaying the degree of the enzymatic activity.

The safety checks such as culture of bacteria and fungi in culturedcells, check for the presence or absence of mycoplasma infection, searchfor endotoxin, etc. are carried out to confirm safety. Thereafter, thecultured cells transformed with the predicted effective dose of thesense oligonucleotide are returned to the patient by intravenous dripinjection. The above procedure is repeated at intervals of several weeksor a few months to consummate the gene therapy.

The dosage of the virus vector is judiciously selected according to thetarget cell. The usually preferred dose may for example be 1×10³cfu-1×10⁸ cfu in terms of virus titer per 1×10⁸ target cells.

It can be adopted an alternative version of the above first method thatcomprises co-cultivating the virus-producer cells having the retrovirusvector harboring the object sense oligonucleotide and the patient'scells to thereby introduce the sense oligonucleotide into the targetcells.

In carrying out the second method (direct method) for gene therapy, itis particularly preferable to perform a preliminary experiment ex vivoto check whether the objective sense oligonucleotide can be actuallyintroduced by the gene transfer method by carrying out PCR of the vectorgene cDNA or in situ PCR or check whether the desired therapeuticeffect, for example elevation of a specific activity or the growth orinhibition of growth of the target cell can be actually achieved byintroduction of the objective sense oligonucleotide. Moreover, when avirus vector is used, it is, of course, of great importance to confirmthe safety of introduction of the sense oligonucleotide in gene therapyby performing a PCR search for proliferative retrovirus and the like,determining the reverse transcriptase activity, or monitoring the coatprotein (env) gene by the RCR technique.

The gene therapy of the invention in Alzheimer's disease, Alzheimer typedementia or Parkinson's disease may for example be a therapy ofneurodegenerative disease which comprises harvesting stem cells or brainnerve cells from the patient, establishing a cultured cell line byenzymatic treatment or the like, introducing the object senseoligonucleotide into the target brain nerve cells utilizing AAV or thelike, carrying out a screening with G418 cells, measuring the amount ofexpression of IL-12 or the like in vivo, giving a radiation treatment,and inoculating the cells into the patient's brain tissue or thetemporal lobe site.

The present invention further provides a pharmaceutical composition orpreparation (a gene therapeutic agent) comprising a senseoligonucleotide transfer vector of the invention or a cell linetransformed with the sense oligonucleotide as an active ingredient in apharmacologically effective amount in combination with a suitablenontoxic pharmaceutical carrier or diluent.

The pharmaceutical carrier that can be utilized in the pharmaceuticalcomposition (pharmaceutical preparation) of the invention includes thosediluents or excipients, e.g. fillers, volume builders, binders,humectants, disintegrators, surfactants, lubricants, etc., which areusually employed depending on the mode of use of such a composition, andthese can be selectively used according to the contemplated unit dosageform of the preparation.

The unit dosage form of the pharmaceutical preparation of the inventionmay be the same as mentioned for the polypeptide preparation of theinvention, and a suitable one can be judiciously selected according tothe therapeutic objective.

The therapeutic and prophylactic method for neurodegenerative diseaseaccording to the invention is now described in detail.

The present invention provides a method for therapy of neurodegenerativediseases, such as Alzheimer's disease, Alzheimer type dementia, brainischemia, Parkinson's disease, and like diseases in which either anexcess or a shortage of the LY6H polypeptide is involved. When LY6Hactivity is excessive, several approaches can be taken. The first methodcomprises administering an inhibitor compound (antagonist) in aneffective amount to inhibit the function of LY6H polypeptide by blockingits binding to a ligand, substrate, receptor, enzyme or the like orinhibiting a secondary signal in combination with a pharmaceuticallyacceptable carrier to thereby improve an abnormal state. An alternativemethod comprises administering a soluble-form LY6H polypeptide capableof binding to a ligand, substrate, enzyme, receptor or the like incompetition with the endogenous LY6H. A typical example of suchcompetitive substance includes a fragment of LY6H polypeptide. Inanother method, a soluble-form LY6H polypeptide capable of binding to aligand in competition with endogenous LY6H can be administered. Atypical example of such competitive substance includes a fragment ofLY6H polypeptide.

In a still another method, expression of the gene coding for endogenousLY6H polypeptide can be inhibited by applying a gene expressioninhibition technique to the LY6H gene product. The known technique ofthis kind includes the use of an internally generated or separatelyadministered antisense sequence [e.g. Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988),O'Connor, J. Neurochem 56: 560 (1991)]. As an alternative method, anoligonucleotide capable of forming a triple helix with the gene can besupplied [e.g. Lee et al., Nucleic Acids Res., 6: 3073 (1979); Cooney etal., Science, 241: 456 (1988); Dervan et al., Science, 251: 1360(1991)]. These oligomers can be administered as such or relatedoligomers may be caused to be expressed in vivo.

For the therapy of abnormal symptoms related to an under-expression ofLY6H and its activity, several methods can be utilized. The first methodcomprises administering a compound capable of activating LY6H (agonist)in a therapeutically effective amount together with a pharmaceuticallyacceptable carrier to a subject to thereby improve the abnormalsymptoms. In another method, the endogenous production of LY6H byrelated cells in the subject can be actuated by gene therapy. Forexample, the polynucleotide of the present invention may be manipulatedso as to be expressed with a defective retrovirus vector as mentionedhereinbefore. Then, this retrovirus expression construct is isolated andintroduced into packaging cells transduced with a retrovirus plasmidvector harboring the RNA encoding the polypeptide of the invention sothat the packaging cells will form infective virus particles containingthe object gene. These producer cells are administered to the subjectfor in vivo manipulation of the cells so that the polypeptide may beexpressed in vivo. For an overview of gene therapy, reference may bemade to Human Molecular Genetics, T. Strachan and A. P. Read, BIOSScientific Publishers Ltd. (1996), Chapter 20—Gene Therapy and OtherMolecular Genetic-based Therapeutic Approaches, inclusive of thespecific references cited therein. An alternative method comprisesadministering a therapeutic dose of LY6H polypeptide in combination witha suitable pharmaceutical carrier.

The cells may for example be formulated in phosphate-buffered saline (pH7.4), Ringer's solution or an intracellular composition injection or insuch a dosage form as can be administered in combination with asubstance conducive to an enhanced gene transfer efficiency, such asprotamine.

The method of administering the above pharmaceutical preparation is notparticularly restricted but a suitable regimen can be establishedaccording to the particular dosage form, the patient's age, sex andother factors, the severity of illness, and the like.

The amount of the active ingredient to be incorporated in thepharmaceutical preparation and the dosage are not particularlyrestricted but each can be liberally selected from a broad rangeaccording to the expected therapeutic benefit, method of administration,duration of treatment, patient background inclusive of age and sex, andother variables.

Generally, the dosage of the retrovirus vector harboring the senseoligonucleotide as a pharmaceutical preparation may for example be about1×10³ pfu through 1×10¹⁵ pfu in terms of retrovirus titer per kilogrambody weight per day.

In the case of cells carrying the sense oligonucleotide forintroduction, the dosage can be properly selected from the range ofabout 1×10⁴ cells/body through 1×10¹⁵ cells/body.

The above preparation can be administered once a day or in a few divideddoses a day, or even intermittently at intervals of 1 or several weeks.Preferably, a substance condusive to an enhanced gene transferefficiency, such as protamine, or a preparation containing the same canbe administered in combination.

When the gene therapy according to the invention is applied to thetherapy of a neurodegenerative disease, it can be performed in asuitable combination with other gene therapies (conjunctive genetherapy) or in combination with a pharmacotherapy utilizing anaqetylcholinesterase inhibitor or the like and/or a rehabilitationtherapy. The gene therapy of the invention can be performed withreference to the NIH guidelines, inclusive of its safety aspect[Recombinant DNA Advisory Committee, Human Gene Therapy, 4, 365-389(1993)].

Furthermore, in accordance with the invention, for the purpose ofdetecting the presence of LY6H gene, it is possible to prepare abiological sample such as blood or serum, optionally extract the nucleicacid, and analyzing it for LY6H gene.

The method of detecting the gene may comprise preparing a DNA fragmentof the gene of the invention and design it so that it may be used in thescreening for LY6H gene and/or its amplification. More specifically, itis possible to construct a DNA fragment having the properties of a probefor plaque hybridization, colony hybridization, Southern blotting,Northern blotting, etc. or a probe for the preparation of a full-lengthor partial DNA of the gene of the invention as amplified by a polymerasechain reaction (PCR) which amplifies a nucleotide sequence with apolymerase. For this purpose, a primer having the same sequence as LY6Hgene is first prepared. Then, this primer is reacted, as a probe forscreening, with a biological sample (nucleic acid sample) to check forthe presence of the particular LY6H gene sequence. The nucleic acidsample may be prepared by any of various techniques facilitatingdetection of the target sequence, such as denaturation, restrictionenzyme digestion, electrophoresis or dot blotting.

As the method for said screening, the use of a PCR technique isparticularly preferred from sensitivity points of view, and thistechnique is not particularly restricted inasmuch as a fragment of thegene of the invention is used as a primer. Thus, It can be utilized thatany of the hitherto-known techniques [Science, 230, 1350-1354 (1985)]and the modified versions of PCR which have been developed of late orwill be developed in the future [Sakaki, Yoshiyuki et al. (ed.), JikkenIgaku (Experimental Medicine), Supplement 8(9) (1990), Yōdosha; Protein,Nucleic Acid, Enzyme: Special Supplement, Kyoritsu Shuppan, 35(7)(1990)].

The DNA fragment for use as the primer is a chemically synthesizedoligo-DNA, and such oligo-DNA can be synthesized using an automated DNAsynthesizer or the like, for example Pharmacia LKB Gene Assembler Plus(Pharmacia). The preferred length of the primer (sense primer orantisense primer) to be synthesized may for example be about 10-30nucleotides. The probe for us in said screening is usually a labeledprobe but may be an unlabeled one, or the detection may be madeaccording to specific binding to a directly or indirectly labeledligand. The suitable label and the method of labeling the probe orligand belong to the prior art. Thus, the prior art label includesradioisotopes, biotin, fluorescent groups, chemiluminescent groups,enzymes, antibodies, etc., which can be taken up through knownprocedures such as nick translation, random priming and kinasetreatment.

The PCR technique to be used for detection may for example be RT-PCR butvarious modifications of the technique which are in routine use in theart can be utilized.

Furthermore, the above assay method can be expediently carried out byutilizing an reagent kit for detecting an LY6H gene in samples.

Therefore, the present invention provides an LY6H gene detection reagentkit comprising a DNA fragment of the gene of the invention.

This reagent kit comprises at least a DNA fragment which hybridizes witha part or the whole of the nucleotide sequence shown in SEQ ID NO:2 orits complementary nucleotide sequence as an essential component and mayoptionally contain other components such as a labeling agent and PCRreagents (for example, Taq DNA polymerase, deoxynucleotidetriphosphates, primers, etc.).

The labeling agent may be a radioisotope or a chemical modifier such asa fluorescent substance but the DNA fragment as such may have beenconjugated with such a labeling agent. This reagent kit may furthercontain a suitable reaction solvent or diluent, standard antibody,buffer, wash solution, reaction stopper solution, etc. which make anassay easier to perform.

The present invention in a further aspect provides a method fordiagnosis of neurodegenerative diseases which comprises using the aboveassay method and a diagnostic agent or diagnostic reagent kit for use inpracticing said method.

By the direct or indirect sequencing of the LY6H genes obtained fromtest samples by utilizing the above method, it is possible to find newLY6H gene-related genes having high homology to the wild-type LY6H gene.

Therefore, the present invention further provides a method of screeningfor human LY6H gene-related genes in samples which comprises performingsaid assay and sequencing of the LY6H genes contained in test samples.

The wild-type LY6H and/or mutant LY6H can be determined by utilizing theprotein encoded by the human LY6H gene of the invention (a polypeptidehaving the amino acid sequence shown in SEQ ID NO:1), a polypeptidehaving an amino acid sequence derived from the sequence shown in SEQ IDNO:1 by the deletion, substitution or addition of 1 or a plurality ofamino acids, a fragment of either of them, or an antibody to any of suchproteins.

Therefore, the invention provides a method of determining ananti-wild-type LY6H and/or mutant LY6H antibody or a method ofdetermining the antigen. By this method, the degree of impairment of thebrain nerve can be detected from a change in wild-type LY6H(polypeptide). Such changes can be detected by the sequencing of LY6H bythe well-established technology described hereinabove, more preferablyby detecting differences in the LY6H polypeptide or the presence orabsence of LY6H polypeptide by the use of said antibody (polyclonal ormonoclonal antibody).

The following is a specific example of determination of said wild-typeand/or mutant LY6H. The anti-LY6H antibody can be used toimmunoprecipitate LY6H polypeptide from a solution containing abiological sample obtained from a human body, such as blood or serum orcan be reacted with the LY6H polypeptide on polyacrylamide gel ofWestern blot or immunoblot. The LY6H polypeptide in a paraffin sectionor frozen tissue specimen can be detected by an immunohistochemicaltechnique using the anti-LY6H antibody. The antibody production andpurification technology are well known in the art and suitabletechniques can be selectively employed.

The preferred technology relevant to the detection of a wild-type LY6Hor a mutant thereof includes enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunoradiometric assay (IRMA) andimmuno-enzymometric assay (IEMA) with a sandwich technique using amonoclonal antibody and/or a polyclonal antibody.

The invention further provides an LY6H ligand or an LY6H receptorexisting in a cell membrane fraction or on a cell surface and havingbinding affinity for LY6H polypeptide. The LY6H receptor can be obtainedby conjugating a labeled LY6H polypeptide in a biological samplecontaining a cell membrane fraction, extracting, isolating and purifyingthe conjugation product and identifying the amino acid sequence of theisolated product. The procedure for preparation and the method ofsequencing this LY6H receptor polypeptide is obvious to one skilled inthe art.

Furthermore, by applying the LY6H receptor or a fragment thereof to ascreening for various drugs, the invention enables selecting out variouscompounds (which react. with the LY6H receptor, inclusive of lowmolecular compounds, high molecular compounds, proteins, proteinfragments, antigens, antibodies, etc.). Preferably, the LY6H receptor asa whole is used. The LY6H receptor polypeptide or fragment thereof foruse in such screening may have been immobilized on a solid matrix or bea free substance in a solution to be transported to the cell surface.

An example of the above pharmacoscreening is a screening system in whichprokaryotic or eukaryotic host cells transformed stably with arecombinant DNA coding for an LY6H polypeptide, or a fragment thereof,are used in, preferably, a competitive binding assay. As an alternative,said host cells, whether in the free form or as immobilized, are used inthe standard binding assay. More particularly, the abovepharmacoscreening may comprise reacting the LY6H receptor polypeptide,or a fragment thereof, with the LY6H polypeptide, or a fragment thereof,in the presence of a candidate drug, to cause formation of a complex anddetecting the degree of inhibition of the complex formation by the abovecandidate drug.

Thus, in accordance with the invention, there can be provided a methodfor pharmacoscreening which comprises contacting a candidate drug withthe LY6H receptor polypeptide, or a fragment thereof and, then,detecting the presence of the resulting complex or the presence of acomplex of the LY6H receptor polypeptide, or a fragment thereof, with aligand by a per se known technique. Furthermore, by assaying LY6Hreceptor activity, it is possible to evaluate whether a candidate drugis capable of antagonizing the LY6H receptor and accordingly may modifythe above-defined LY6H activity, i.e. may be able to modulate growth ofneurons, or modulate protein-protein conjugation or complex-formingactivity. In such a competitive binding assay, the LY6H receptorpolypeptide, or a fragment thereof, is labeled. When the free LY6Hreceptor polypeptide or fragment thereof is separated from theprotein-protein complex and the labeling amount of the free(non-complex-forming) substance is measured, the measured value servesas a yardstick of the binding of the test factor to the LY6H receptor.The measured value serves also as a measure of inhibition of the bindingof the LY6H receptor to the LY6H polypeptide. By analyzing a smallpeptide (pseudopeptide) of the LY6H polypeptide in this manner, thecandidate drug can be assayed as a substance having LY6H receptorantagonizing activity.

Another protocol for pharmacoscreening in accordance with the inventionis that of screening for a compound having an adequate binding affinityfor the LY6H receptor polypeptide. Briefly, this procedure comprisessynthesizing a large number of different test peptide compounds on asolid support such as the surface of a plastic pin or other material,reacting the test peptide compounds with the LY6H receptor polypeptideand, after washing, detecting the binding reaction products of LY6Hreceptor polypeptide by a known method [e.g. PCT patent publication No.WO 84-03564]. The purified LY6H receptor can be directly coated on theplate to be used in said pharmacoscreening procedure. The antibody maybe captured with a non-neutralizing antibody against the polypeptide andthe LY6H receptor polypeptide be immobilized on a solid phase.

The invention is further directed to the use of a competitivepharmacoscreening assay. For the binding to the LY6H receptorpolypeptide, or a fragment thereof, a neutralizing antibody capable ofspecific binding to the LY6H receptor polypeptide is caused to competewith the candidate compound. By such a competitive reaction with theneutralizing antibody, the presence of any peptide having one or moreantigenic determinants of the LY6H receptor polypeptide can be detected.

As a further method for drug screening, the LY6H polypeptide of theinvention or the LY6H gene-product of the invention can be used in thescreening for compounds which activate (agonists) or inhibit(antagonists or inhibitors) the activity of the LY6H polypeptide or LY6Hgene product.

By using the LY6H polypeptide or LY6H gene product of the invention,agonists or antagonists can be identified from cells, cell-freepreparations, chemical libraries and naturally-occurring compositions.These agonists or antagonists may be natural or modified substrates,ligands, enzymes or receptors of the LY6H polypeptide of the inventionor structural or functional copies of the polypeptide of the invention[Coligan et al., Current Protocols in Immunology, 1(2), Chapter 5(1991)].

In situ hybridization studies revealed the expression of LY6H gene ofthe invention in various tissues of the human normal brain, atparticularly high levels in the hippocampus and entorhinal cortex whichare usually severely impaired in Alzheimer patients, and its expressionlevel has been found to be considerably depressed in the temporal lobeinclusive of the hippocampus and entorhinal cortex of patients withAlzheimer's disease. It is, therefore, very likely that this gene isassociated with the onset and progression of said disease.

Therefore, an agonist or antagonist of this LY6H protein or an LY6H geneproduct is expected to find application as a therapeutic or prophylacticdrug for neurodegenerative diseases such as Alzheimer's disease,Alzheimer type dementia, brain ischemia and Parkinson's disease.

Compounds obtainable by the screening for candidate drugs for said LY6Hgene-related diseases have the functions of the protein of the invention(the expression product of the gene of the invention), such as neuronalsurvival-supporting action, nerve elongating action, nerve regeneratingaction, neuroglia-activating action, etc. in the central and other nervesystems and brain mnemonic (memory-forming) action, among otherphysiological actions, and, therefore, can be used as a therapeutic orprophylactic drug for various neurodegenerative diseases such asAlzheimer's disease, Alzheimer type dementia, brain ischemia andParkinson's disease. Thus, the proteins of the invention (inclusive ofthe gene expression products, partial peptides thereof, and saltsthereof) are of use as reagents for the screening for compounds whichpromote the functions of the protein of the invention.

The invention provides a method of screening for compounds which promotethe functions of the protein of the invention (hereinafter each referredto sometimes as a functional enhancer of the protein of the invention).More particularly, the invention provides (a) a method of screening fora functional enhancer of the protein of the invention which comprisescontacting (1) the protein of the invention with nerve cells or a nervetissue on one hand and (2) the protein and a test compound with saidnerve cells or tissue on the other hand and comparing the results and(b) a method of screening for a functional enhancer of the protein ofthe invention which comprises administering (1) the protein of theinvention to a vertebrate on one hand and (2) the protein of theinvention and a test compound to the vertebrate on the other hand andcomparing the results.

More particularly, in the above screening method (a), a physiologicalactivity in the central or other nervous systems, such as neuronalsurvival-supporting activity, nerve elongating activity, nerveregenerating activity or neuroglia-activating activity, is measuredunder the above conditions (1) and (2) and the results are compared. Inthe screening method (b), the mnemonic (memory-forming) activity in thebrain, for instance, is measured under said two conditions (1) and (2)and the results are compared.

The nerve cells (neurous and neuroglia) for use in the above screeninginclude neuroblastoma. cells, glioma cells, and their hybridoma cells(e.g. N18TG-2, IMR-32, GOTO (e.g. GOTO-P3), NB1, C6BU-1, U251, KNS42,KNS81 and NG108-15 cells, and PC12 cells having a potency ofdifferentiation to nerve cells). The nerve tissue which can be usedincludes the mouse neuroepithelial cell, rat hippocampus primary culturecell, fetal mouse culture Prukinje cell, and mouse dorsal root ganglia.The test compound includes peptides, proteins, nonpeptide compounds,synthetic compounds, fermentation. products, cell extracts, plantextracts, animal tissue extracts, and plasma. These compounds may benovel compounds or known compounds.

In carrying out said screening method (a), the protein of the invention(inclusive of a partial peptide thereof or a salt thereof) is dissolvedor suspended in a screening buffer to prepare a sample of the protein ofthe invention. The buffer may be any buffer solution that does notinterfere with the contact between the protein of the invention and thenerve cell or tissue (e.g. phosphate buffer, Tris-HCl buffer, etc. at pHabout 4-10, preferably pH about 6-8). The duration of contact is usuallyabout 1-10 days, preferably about 7-10 days. The contact temperature isusually about 37° C. The activities of the protein of the invention inthe central or other nervous systems, such as neuronalsurvival-supporting. activity, nerve elongating activity, nerveregenerating activity, and neuroglia-activaing activity, can bedetermined by the routine methods such as visual assessment of axonalelongation, measurement of intracellular Ca²⁺ concentration, and thelike.

Any test compound promoting any of said physiological activities, suchas neuronal survival-supporting activity, nerve elongating activity,nerve regenerating activity, neuroglia-activating activity, by at leastabout 20%, preferably not less than about 30%, more preferably notless.than about 50%, still more preferably not less than about 70%,under the above-mentioned condition (2) as compared with the condition(1) can be selected as a functional enhancer of the protein of theinvention.

In carrying out the above screening method (b), the protein of theinvention, alone or in combination with the test compound, isadministered to test animals by intravenous, subcutaneous orintramuscular injection or orally. The dosage of the protein of theinvention for oral administration is,generally about 0.1-100 mg/day,preferably about 1.0-50 mg/day, more preferably about 1.0-20 mg/day, permammal (based on 50 kg body weight). The parenteral dose should beselected according to the recipient and the method of administration butit is preferable to administer about 0.01-30 mg/day, preferably about0.1-20 mg/day, more preferably about 0.1-10 mg/day, per mammal (50 kgbody weight) by the intravenous route.

Test animals include such mammals as man, monkey, chimpanzee, mouse,rat, rabbit, sheep, swine, bovine, horse, cat and dog and fish (e.g.carp, salmon, herring, rainbow trout, goldfish, etc.).

The mnemonic (memory-forming) activity of the protein of the inventionin the brain can be assayed in accordance with, for example, a watermaze test protocol [Morris, R. G. M., J. Neurosci. Meth., 11, 47-60(1984)]. Any test compound promoting the above mnemonic effect by notless than about 20%, preferably not less than 50%, more preferably notless than 70%, under said condition (2) as compared with said condition(1) is of use as a functional enhancer of the protein of the invention.

The screening kit as a further embodiment of the invention contains theprotein of the invention (inclusive of the expression product of thegene, a partial peptide thereof, and any salt of either of them) as anessential component. A kit consists of the following components 1-4 isan example of the screening kit of the invention.

-   Component 1: Hanks solution as assay buffer-   Component 2: Protein standard (protein of the invention or a salt    thereof)-   Component 3: Nerve cells or a nerve tissue (a culture of said nerve    cells or nerve tissue in a 24-well plate, 10⁴ cell/well, as grown    using Eagle's MEM, Hanks solution under 5% CO₂ at 37° C.)-   Component 4: An inverted microscope for observation

The screening with the above screening kit can be carried out asfollows.

[Method]

The number per field of vision of axonal elongation-positive cells inthe well containing the test compound is counted and compared with thenumber of axonal elongation-positive cells in the control (testcompound-free) well and the difference is statistically tested.

The compound or salt obtained by the screening method or with thescreening kit in accordance with the invention is a member selected fromthe above-mentioned class consisting of peptides, proteins, nonpeptidecompounds, synthetic compounds, fermentation products, cell extracts,plant extracts, animal tissue extracts, etc. and is a compound capableof promoting the function of the protein of the invention. The compoundthat promotes the functions of the protein of the invention as such mayshow physiological activities such as neuronal survival-supportingactivity, nerve elongating activity, nerve regenerating activity,neuroglia-activating activity, etc. and thereby promote the function ofthe protein of the invention or the like additively or synergisticallyor, although not showing such physiological activities by itself, maypromote the function of the protein of the invention. Examples of thesalts of the compound include salts with physiologically acceptablebases (e.g. alkali metals) or acids (e.g. organic acids, inorganicacids). Particularly preferred are physiologically acceptable acidaddition salts, such as salts with inorganic acids (e.g. hydrochloricacid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids(e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleicacid, succinic acid, tartaric acid, citric acid, malic acid, oxalicacid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The compound or salt which promotes the function of the protein of theinvention is of value as a safe, low-toxicity therapeutic-prophylacticdrug for various neurodegenerative diseases such as Alzheimer's disease,Alzheimer type dementia, brain ischemia and Parkinson's disease.

The above screening procedure involves the use of cells which expressthe LY6H polypeptide on the cell surface or respond to the protein ofthe invention. Among such cells are cells derived from mammaliananimals, yeasts, Drosophilia and E. coli. The cells which express theLY6H polypeptide (or the cell membrane having the expressed polypeptide)or respond to the LY6H polypeptide is contacted with the test compoundto observe the stimulation or inhibition of binding or functionalresponse. Then, LY6H activity of cells contacted with the candidatecompound is compared with that of similar cells not contacted.

The above assay can be carried out by detecting adhesion to cellsharboring the LY6H polypeptide using a label directly or indirectlycoupled to a candidate compound or in an assay system utilizing acompetition with a label-competitive substance. In this manner, thebinding of the candidate compound can be easily tested. Furthermore,using a detection system suited to cells bearing the LY6H polypeptide insuch assays, it may be tested whether the candidate compound willproduce a signal ascribable to activation of the LY6H polypeptide. Theactivation inhibitor is generally assayed in the presence of a knownagonist and the effect of the candidate compound on the activation dueto the agonist is observed. The assay may comprise a simple procedurecomprising mixing the candidate compound with a solution containing theLY6H polypeptide to form a mixture, determining the LY6H activity in themixture, and comparing the LY6H activity of the mixture with a standard.

The low molecular compound (agonist or antagonist) which binds to theLY6H protein can be obtained by a screening with BIACORE 2000, forinstance [Markgren, P. O., et al., Analytical Biochemistry, 265, 340-350(1998)].

In accordance with the invention, for the purpose of developing a moreactive or stabilized LY6H polypeptide derivative or a drug whichenhances or blocks the function of the LY6H polypeptide in vivo, it ispossible to construct a biologically active polypeptide or a structuralanalog thereof for interaction, such as an LY6H agonist, LY6Hantagonist, LY6H inhibitor or the like. The structural analog mentionedabove can be obtained, for example, by determining the three-dimensionalstructure of a complex of LY6H polypeptide with another protein by X-raycrystallography, computer modeling or a combination of such techniques.Information on the structure of a structural analog can also be acquiredby polypeptide modeling based on the structures of homologous proteins.

To obtain said more active or stabilized LY6H polypeptide derivative,analysis by alanine scan can. be employed. This method comprisessubstituting Ala for each amino acid residue to assess the influence ofsubstitution on peptide activity. Thus, as each amino acid residue of apeptide is thus analyzed, the region of importance to the activity orstability of the peptide is determined. By this method, it is possibleto design a more active or stable LY6H polypeptide derivative.

It is also possible to isolate the target-specific antibody selected bythe functional assay and analyze its crystal structure. As a rule, bythis approach, the pharmacore providing a basis for subsequent drugdesign is obtained. By producing an anti-ideotypic antibody to thefunctional pharmacologically active antibody, it is possible to identifyand isolate a peptide from a chemically or biologically generatedpeptide bank. Therefore, it is predictable that the selected peptide mayalso serve as a pharmacore.

In this manner, it is possible to design and develop drugs havingimproved or stabilized LY6H activity or acting as inhibitors, agonistsor antagonists of LY6H activity.

Evaluation of such a drug can be made by titrating its effect onneuronal survival using primary culture hippocampal neurons [Japan. J.Pharmacol, 53, 221-227 (1990)] or investigating its effect onneurodegenerative lesions in Alzheimer model animals such as mutantβ-amyloid precursor protein gene or mutant presenilin 1 gene transgenicmice [Nature, 373, 523-527 (1995): Nature Med., 5, 560-564 (1999)].

The compound thus obtained can be used not only as a drug forAlzheimer's disease but also as a therapeutic drug for cerebralinfarction and other neurodegenerative diseases.

Furthermore, in accordance with the invention, by constructing LY6Hgene-bearing knockout mice (transgenic mice with LY6H knockoutbackgrounds), it is possible to ascertain which site or sites of thenucleotide sequence of the LY6H gene have influences on said multipleLY6H activities in vivo, that is to say what functions the expressionproducts of LY6H gene and of a modified LY6H gene have in vivo.

This method is a technique to intentionally modify the geneticinformation of a living thing by utilizing homologous recombinant genes,and includes a method using mouse embryonic stem cells (ES cells) as anexample [Capeccchi, M. R., Science, 244, 1288-1292 (1989)].

The method of constructing said mutant mice is by now a routinetechnology for those skilled in the art, and mutant mice can be easilyconstructed by applying a human wild-type Ly6H gene or a mutant LY6Hgene to a modified version of the above technology [Noda, Testuo (ed.):Jikken Igaku (Experimental Medicine), Supplement, 14(20) (1996),Yodosha]. Therefore, by utilizing this technique, it is possible todesign and develop drugs having improved or stabilized LY6H activity orinhibitors, agonists, and antagonists of LY6H activity.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagrammatic representation of Northern blots showing thepattern of expression of LY6H gene in various sites of the brain of apatient with Alzheimer's disease.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are intended to illustrate the invention infurther detail.

EXAMPLE 1

(1) Cloning and DNA Sequencing of Human LY6H Gene

The mRNA extracted from the human fetal brain was purchased fromCLONTECH Laboratories and used as the starting material. From this mRNA,a cDNA was synthesized and ligated into the vector λZAPII (Stratagene)to construct a cDNA library (Otsuka GEN Research Institute, OtsukaPharmaceutical Co.). Using the in vivo excision method [in vivoexcision: Short, J. M., et al., Nucleic Acids Res., 16, 7583-7600(1988)], colonies of Escherichia coli bearing the human gene were formedon agar medium and randomly picked up to register the human gene-bearingE. coli clones in a 96-well microplate. These clones were stored at −80°C.

Then, each registered clone was cultured in 1.5 ml of LB mediumovernight and the DNA was extracted and purified using an automaticplasmid extractor PI-100 (Kurabo). The contaminated E. coli RNA wasdecomposed with RNase and removed. Finally, 30 μl of a DNA solution wasprepared and using a 2 μl portion, the approximate DNA size and amountwere checked by the minigel method. A 7 μl portion was used for asequencing reaction and the remaining 21 μl was stored as plasmid DNA at4° C. By this method, a cosmid which can also be used as a probe forFISH (fluorescence in situ hybridization) described below can beextracted by a minor modification of the program.

Then, a dideoxy terminator reaction of Sanger et al. using T3, T7 or asynthetic oligonucleotide primer [Sanger, F., et al., Proc. Natl. Acad.Sci., USA., 74, 5463-5467 (1977)] or a cycle sequencing reaction[Carothers, A. M., et al., Bio. Techniques, 7, 494-499 (1989)] which isthe dideoxy terminator reaction plus PCR was carried out. These aretechniques for chain extension with termination specific to 4 kinds ofbases using a small amount (about 0.1-0.5 μg) of plasmid DNA as thetemplate.

Using an FITC (fluorescein isothiocyanate)-labeled primer as thesequence primer, about 25 cycles of reaction using Taq polymerase werecarried out. Of the fluorescence-labeled DNA fragment, the sequence ofabout 400 nucleotides from the 5′-end of the cDNA was determined withthe automatic DNA sequencer ALF™ DNA Sequencer (Pharmacia).

The 3′-nontranslated region is high in heterogeneity among genes andsuited for differentiation of individual genes. Therefore, sequencing ofthe 3′-end region was also performed in some cases.

The huge nucleotide sequence information generated with the DNAsequencer was transmitted to the 64-bit computer DEC3400 forcomputerized homology analysis. This homology analysis was carried outby a database (GenBank, EMBL) search according to UWGCG's FASTA Program[Pearson, W. R. and Lipman, D. J., Proc. Natl. Acad. Sci., USA., 85,2444-2448 (1988)].

Fujiwara et al. describe in detail about the above method of analysisfor a human fetal brain cDNA library [Fujiwara, T., et al., DNA Res., 2,107-111 (1991)].

The ESTs (expressed sequence tags: partial DNA sequences of theexpressed gene fragment) randomly selected from the human fetal braincDNA library constructed as above were then sequenced.

The clone designated GEN-425D01 in the GenBank/EMBL sequence searchaccording to the FASTA Program was found to be highly homologous to thegene coding for the mouse Ly6 family protein.

Using a double-stranded DNA inserted into a vector (pBluescript vector;Stratagene) as a template and a synthetic oligonucleotide as a primer,the nucleotide sequence of the cDNA inclusive of the whole coding regionof the above clone was determined by Sanger's dideoxy chain terminationmethod.

Sequencing with ABIPRISMTM377 automatic DNA sequencer revealed that thecDNA sequence of the clone obtained above contained a deduced amino acidcoding region of 420 bases and the amino acid sequence encoded therebyhad 140 amino acid residues. The nucleic acid sequence of thefull-length cDNA clone was composed of 854 nucleotides. The fullsequence is shown in SEQ ID NO:3; the nucleotide sequence of the openreading frame is shown in SEQ ID NO:2; and the deduced amino acidsequence encoded by said nucleotide sequence is shown in SEQ ID NO:1.

The amino acid sequence of the human LY6H protein was compared with thesequences of other Ly6 family proteins, and the nucleotide sequenceconserved in the amino acid translation initiation region [Kozak, M., J.Biol. Chem., 266, 19867-19870 (1991)] was compared with the 5′-region ofthe human LY6H gene. The initiation codon thus determined was located inthe position 99-101, which is the second ATG triplet, of the nucleotidesequence shown in SEQ ID NO:3. Moreover, the polyadenylation signal(AATAAA) was located in the position 832-837 of the same nucleotidesequence.

(2) Northern Blot Analysis

To define the expression profile of LY6H in tissues, a Northern blotanalysis was performed using various human tissues.

In the Northern blot analysis, Human MTN (Multiple-Tissue Northern) BlotI and II (CLONTECH) were used.

The cDNA fragment was amplified by PCR using a primer set of T3 and T7promoter sequences.

The PCR amplification product of said GEN-425D01cDNA clone was labeledwith [³²P]-dCTP (Random Primed DNA Labeling Kit, Boehringer MannheimGmbH) for use as a probe.

The blot containing the amplification product was prehybridized (underconditions according to the product protocol) and, then, subjected tohybridization according to the product protocol.

The hybridization was performed at 65° C. overnight in a solutioncomposed of 1 M NaCl/50 mM Tris-HCl (pH 7.5)/2×Denhardt's solution/10%dextran sulfate/1% SDS solution (containing 100 μg/ml denatured salmonsperm DNA). After wash twice with 2×SSC/0.1% SDS at room temperature,the product was washed once with 0.1×SSC/0.1% SDS at 65° C. for 40minutes. The filter was exposed against X-ray film (Kodak) at −70° C.for 18 hours.

The above test was performed using the following adult human tissues:brain, pancreas, testis, small intestine, colon, thymus, prostate,ovary, heart, placenta, lung, liver, skeletal muscle, kidney, spleen,testis and peripheral blood leukocyte. As a result, transcripts of about1 kb showing homology to LY6H were observed in the brain, pancreas,testis, small intestine, colon, thymus, prostate and ovary, particularlyhigh in the brain.

(3) Localization of the Gene on Chromosome by FISH Using Cosmid Clones

FISH for chromosomal localization was carried out using 0.5 μg of eachcosmid DNA as a probe in accordance with the known method [Takahashi, E.et al., Hum. Genet., 86, 14-16 (1990)]. It was caught FISH signals byProvia 100 film (Fuji, ISO 100) or CCD Camera System (Applied ImagingCyto Vision).

As a result, the human LY6H gene was found to be located on q24.3 ofchromosome 8. Thus, GEN-425D01 was mapped on the chromosome band 8q24.3.

The antibodies against proteins belonging to the Ly6 family have beenutilized in the purification of blood stem cells as a target of genetherapy [van de Rijn, M., et al., Proc. Natl. Acad. Sci., USA., 86,4634-4638 (1989)], studies on the differentiation of blood cells [van deRijn. M., et al., Proc. Natl, Acad. Sci., USA., 86, 4634-4638 (1989);Classon, B. J. and Coverdale, L., Proc. Natl. Acad. Sci., USA., 91,5296-5300 (1994)], activation of immune cells [Malek, T. R., et al., J.Exp. Med., 164, 709-722 (1986)], inhibition of production of activeimmune cells [Haque, A., et al., Immunology, 69, 558-563 (1990)], andthe like, and have also been found to have antitumor effects [Lu, L., etal., J. Immunol., 142, 719-725 (1989)]. The human LY6H gene provided inthe present example enables detection of the expression of the gene invarious tissues, production of the human LY6H protein by geneticengineering techniques, and construction of an antibody by utilizing thegene, hence enabling said purification of blood stem cells, researchinto the differentiation of blood cells, activation of immune cells,inhibition of activation of immune cells, and therapy of tumors.

Furthermore, the LY6H expressed at a high level in the brain enables aresearch into the differentiation of nerve cells, activation of neurons,and therapy of neural and mental diseases.

Screening for compounds with the human LY6H protein as the target isalso made possible and the compounds thus obtained are as useful as theanti-human LY6H protein antibody.

EXAMPLE 2

(1) Northern Blot Analysis in the Brain Tissues of a Patient withAlzheimer's Disease

Northern blot analysis was performed in accordance with Example 1 (2).

To investigate the expression of the LY6H gene in the brain tissues ofpatients with Alzheimer's disease, Northern blot analysis was made usingthe brain tissues of an Alzheimer patient and normal human braintissues.

Northern blotting was performed using the human normal brain blot II andhuman Alzheimer blot II (both Invitrogen) and the LY6H gene expressionin the various brain tissues, namely the frontal lobe, temporal lobe,parietal lobe, occipital lobe, pons, thalamus and corpus callosum, wascompared between normal and Alzheimer brains.

The results are shown in FIG. 1.

As was pointed out in Example 1, the LY6H gene is expressed at a highlevel in the brain. The above analysis revealed that, while theexpression of the gene was confirmed in various tissues of the humannormal brain, the gene was expressed at particularly high levels in thetemporal lobe inclusive of the hippocampus and entorhinal cortex whichare known to be impaired severely in patients with Alzheimer's diseasewhile marked decreases were found in the temporal lobe inclusive of thehippocampus and entorhinal cortex in the patient with Alzheimer'sdisease, indicating that it is very likely that the gene is involved inthe onset and progression of this disease.

Therefore, the LY6H gene sense strand, LY6H expression product, and LY6Hprotein are expected to find application as therapeutic drugs forAlzheimer's disease, Alzheimer type dementia, brain ischemia andParkinson's disease.

Furthermore, agonists and antagonists of LY6H protein are also expectedto be of use as therapeutic drugs for Alzheimer's disease and otherdiseases.

EXAMPLE 3

(1) Construction of an LY6H expression vector The LY6H cDNA obtained byin vivo excision method is cleaved with MvlI and XhoI to give an about800-base fragment. This fragment, containing the entire coding region ofthe LY6H gene shown in SEQ ID NO:1, is ligated to the EcoRV/XhoI-cleavedpAc5.1/V5-HisA (Invitrogen) to construct an expression vector (pAC/LY6Hexpression vector).(2) Expression and Purification of the Active Ingredient Protein of theInvention

The pAC/LY6H expression vector DNA and pCoHYGRO vector (Invitrogen) DNAare admixed in a ratio of 19:1 and introduced into fruit fly (Schneider2) cells by calcium phosphate transfection. After the cells are culturedin 10% fetal calf serum-DES expression medium (Invitrogen) at 23° C. for48 hours, 300 μg/ml of hygromycin (Hygromycini B, Boehringer Mannheim)is added to the culture and the selection of drug-resistant cell clonesis performed for 2 weeks. A stable transformant is subjected tostationary culture at a concentration of 5×10⁶ cells/ml using 20 Falcon5000 culture flasks (Becton Dickinson) containing 20 ml of 10% fetalcalf serum-DES expression medium (Invitrogen) and the cultured cells areharvested. After washing twice with phosphate-buffered saline (PBS), thecells are suspended in PBS containing 2% bovine serum albumin and 0.5U/ml of phosphatidyl-inositol-specific phospholipase C (PIPLC) andcultured at 37° C. for 1 hour. From the supernatant of the culture, theobjective protein can be purified by ion exchange column chromatographyor the like.

(3) Isolation and Culture of Hippocampal Neurons

The whole brain is aseptically isolated from fetal SD rats on embryonicday 18 and the hippocampus is excised. The excised tissue is cut to thinslices with a surgical knife and incubated for enzymatic treatment inPBS containing 0.25% trypsin and 0.002% DNase I at 37° C. for 20minutes. After the enzymatic reaction is stopped by adding fetal calfserum, the aspiration-ejection of the cell digest with a pipette havinga plastic tip is repeated 3 times to disperse the cells. The celldispersion is passed through a filter consisting of 2 stacked sheets oflens paper to remove the undigested tissue and centrifuged at 1000 rpmfor 5 minutes. The cells are washed with DMEM (Gibco) and seeded on apoly-L-lysine (Sigma)-coated 96-well plate containing 10% FCS-DMEM at afinal concentration of 2×10⁵ cells/cM².

(4) Treatment with the Active Ingredient Protein of the Invention

The above cells are cultured for 24 hours and after the culture mediumis changed to 1% N₂ Supplement (Gibco)-DMEM, the active ingredientprotein of the invention as prepared under (2) is added (the inventiongroup).

For comparison, the active ingredient protein of the invention isheat-treated in a boiling water bath for 5 minutes and added (the boiledprotein group).

(5) Evaluation of Hippocampal Neuronal Survival

The cells (culture) in each group as prepared under (4) are cultured for72 hours. Then, the hippocampal neuronal survival-supporting effect ofthe active ingredient protein of the invention can be evaluated by theMTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay. This MTT assay may be performed using Promega's “CellTiter 96”Assay System, for instance.

(6) Isolation and Culture of Midbrain Neurons

The whole brain is aseptically isolated from fetal SD rats on embryonicday 14 and the ventral midbrain is excised. The tissue is cut to thinslices with a surgical knife and incubated for enzymatic treatment inphosphate-buffered saline (PBS) containing 0.25% trypsin and 0.002%DNase I at 37° C. for 20 minutes. After the enzymatic reaction isstopped by adding fetal calf serum, the aspiration-ejection of the celldigest with a pipette having a plastic tip is repeated 3 times todisperse the cells. The cell dispersion is passed through a filterconsisting of 2 stacked sheets of lens paper to remove the undigestedtissue and centrifuged at 1000 rpm for 5 minutes. The cells are washedwith DMEM/F12 (Gibco) and seeded on a poly-L-lysine-coated 96-well platecontaining 10% FCS-DMEM/F12 at a final concentration of 3×10⁵ cells/cm².

(7) Treatment with the Active Ingredient Protein of the Invention

The cells prepared under (6) are cultured for 24 hours and after theculture medium is changed to 1% N₂ Supplement (Gibco)-DMEM/F12, theactive ingredient protein of the invention as prepared under (2) isadded (the invention group).

For comparison, the active ingredient protein of the invention isheat-treated in a boiling water bath for 5 minutes and added (the boiledprotein group).

(8) Evaluation of Midbrain Neuronal Survival Supporting Effect

The cells (culture) in each group as prepared under (7) are cultured for72 hours. Then, the midbrain neuronal survival-supporting effect of theactive ingredient protein of the invention can be evaluated by the MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.This MTT assay may be performed using Promega's “CellTiter 96” AssaySystem, for instance.

(9) Evaluation of Dopaminergic Neuronal Survival-supporting Effect

The cells (culture) in each group as prepared under (7) are cultured for72 hours and, then, fixed by allowing them to stand in 4%paraformaldehyde-PBS at room temperature for 15 minutes. Thereafter,using 1% Triton X100/PBS, it is passed through a membrane.

To prevent nonspecific binding of the antibody, the cells are incubatedin 10% goat serum-PBS for 1 hour and, then, using an anti-tyrosinehydroxylase polyclonal antibody (Chemicon; diluted 1000-fold with PBS),the cells are incubated at 4° C. for 16 hours. After the antibodysolution is removed, the cells are washed with PBS and, withperoxidase-labeled dextran polymer-conjugated goat anti-rabbitimmunoglobulin (Dako) added, the cells are incubated at room temperaturefor 1 hour.

The tyrosine hydroxylase-positive cells can be detected by the colorreaction using diaminobenzidine as the substrate. Using the number oftyrosine hydroxylase-positive cells as a marker, the dopaminergicneuronal survival-supporting effect can be evaluated.

INDUSTRIAL APPLICABILITY

The present invention provides a novel brain-specific gene and a proteinencoded thereby and, by utilizing them, technologies of value to thepurification of blood stem cells, research into the differentiation ofblood cells, activation of immune cells, inhibition of production ofactive immune cells, and therapy of tumors can be provided. Also, thepresent invention provides novel genes having physiologic activitiessuch as brain neuronal survival-supporting activity, nerve elongatingactivity, nerve regenerating activity, neuroglia-activating activity andbrain memory-forming activity.

In view of the marked depression of its expression level in the temporallobe of the brain of patients with Alzheimer's disease, the gene of theinvention is considered to inhibit neurodegenerative changes of thetissue, thus being of use as a gene therapy drug. Moreover, theexpression product of the gene of the invention finds application as aprophylactic and therapeutic drug for such neurodegenerative diseases.

1. An isolated polynucleotide consisting of a nucleotide sequence codingfor a polypeptide consisting of the amino acid sequence of SEQ ID NO:1,or a complementary strand thereof.
 2. An expression vector comprisingthe polynucleotide according to claim
 1. 3. A host cell comprising theexpression vector according to claim
 2. 4. An isolated polynucleotideconsisting of an open reading frame consisting of the nucleotidesequence of SEQ ID NO:2.
 5. An isolated polynucleotide consisting of thenucleotide sequence of SEQ ID NO:3.
 6. An expression vector comprising apolynucleotide according to claim 4 or
 5. 7. A host cell comprising theexpression vector according to claim
 6. 8. A method of producing apolypeptide consisting of the amino acid sequence of SEQ ID NO:1,comprising: (a) culturing the host cell of claim 6 under conditionspromoting expression of said polypeptide, and (b) recovering saidpolypeptide from the cell culture.